UNIVERSITY  OF  CALIFORNIA  PUBLICATIONS 

COLLEGE  OF  AGRICULTURE 

AGRICULTURAL  EXPERIMENT  STATION 

BERKELEY,  CALIFORNIA 


THE  INTRADERMAL  TEST  FOR 

TUBERCULOSIS  IN  CATTLE 

AND  HOGS 


BY 

CLARENCE  M.  HARING 

ASSISTED     BY 

RALPH  M.  BELL 


BULLETIN  No.  243 

Berkeley,  CaL,  March,  1914 


UNIVERSITY   OF  CALIFORNIA  PRESS 
BERKELEY 

1914 


Benjamin  Ide  Wheeler,  President  of  the  University. 

EXPERIMENT  STATION  STAFF 
HEADS   OF   DIVISIONS 

Thomas  Forsyth  Hunt,  Director. 

Eugene  W.  Hilgard,  Agricultural  Chemistry  (Emeritus). 

Edward  J.  Wickson,  Horticulture. 

Herbert  J.  Webber,  Director  Citrus  Experiment  Station;  Plant  Breeding. 

Hubert  E.  Van  Norman,  Vice-Director;  Dairy  Management. 

William  A.  Setchell,  Botany. 

Myer  E.  Jaffa,  Nutrition. 

Robert  H.  Loughridge,  Soil  Chemistry  and  Physics  (Emeritus). 

Charles  W.  Woodworth,  Entomology. 

Ralph  E.  Smith,  Plant  Pathology. 

J.  Eliot  Coit,  Citriculture. 

John  W.  Gilmore,  Agronomy. 

Charles  F.  Shaw,  Soil  Technology. 

John  W.  Gregg,  Landscape  Gardening  and  Floriculture. 

Bernard  A.  Etcheverry,  Irrigation. 

Frederic  T.  Bioletti,  Viticulture  and  Enology. 

Warren  T.  Clarke,  Agricultural  Extension. 

John  S.  Burd,  Agricultural  Chemistry. 

Charles  B.  Lipman,  Soil  Chemistry  and  Bacteriology. 

Clarence  M.  Haring,  Veterinary  Science  and  Bacteriology. 

Ernest  B.  Babcock,  Genetics. 

Gordon  H.  True,  Animal  Husbandry. 

James  T.  Barrett,  Plant  Pathology. 

Fritz  W.  Woll,  Animal  Nutrition. 

Burr  B.  Pratt,  Pomology. 

William  G.  Hummel,  Agricultural  Education. 

Frank  L.  Peterson,  Farm  Mechanics. 

David  N.  Morgan,  Assistant  to  the  Director. 

Mrs.  D.  L.  Bunnell,  Librarian. 


DIVISION   OF  VETERINARY   SCIENCE 

Clarence  M.  Haring  Walter  J.  Taylor 

Chester  L.  Roadhouse  James  F.  Mitchell 

Fred  M.  Hayes  Ralph  M.  Bell 


[94] 


CONTENTS 


PAGE 

Foreword    : 97 

General  Conclusions  Kegarding  the  Intradermal  Method  of  Testing  Cattle.     98 

Present  Status  of  the  Intradermal  and  Ophthalmic  Tests  in  the  United  States.  100 

Local   Tuberculin   Eeactions   and  Their  Use  for  Diagnosis   in   Human   and 

Veterinary  Practice   101 

Technic  of  the  Intradermal  Method  102 

Kind  and  Strength  of  Tuberculin  for  Intradermal  Use  104 

Precautions  against  the  Deterioration  of  Tuberculin  105 

Lack  of  Uniformity  in  Tuberculin  106 

Alcoholic  Precipitated  Tuberculin   107 

Koch's  Old   Tuberculin   109 

Autopsy  Notes  Ill 

The  Local  Reaction  to  the  Intradermal  Injection  in  Cattle  117 

The  Thermal  Reaction  to  the  Intradermal  Injection  in  Cattle  118 

Accuracy  of  the  Intradermal  Compared  with  the  Subcutaneous  Method 122 

Results  of  Retests  127 

Description  of  Cases  which  Reacted  to  the  Intradermal  but  did  not  React 

to  the  Subcutaneous  Test  129 

Discussion    of   Cases   which   Reacted    Thermally   to   the    Subcutaneous,   but 

Showed  no  Local  Reaction  to  the  Intradermal  Test  133 

Comparative  Advantages  of  Intradermal  and  Ophthalmic  Methods  in  Test- 
ing Cattle   134 

Use  of  the  Intradermal  Tuberculin  Test  on  Swine  135 

Conditions  under  which  the  Intradermal  Test  is  Preferable  136 

Conditions  under  which  the  Intradermal  Test  is  Unreliable  137 

Answers  to  Certain  Objections  to  the  Intradermal  Method  138 

Summary    140 

References  143 

Acknowledgment 145 


[95] 


96 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


Table 

1. 

Table 

2. 

Table 

3. 

Table 

4. 

Table 

5. 

Table 

6. 

Table 

7. 

Tables  A 

TABLES 

PAGE 

Results  from  the  Intradermal  Use  of  Various  Solutions  of  Tuber- 
culin      110 

Results  of  Intradermal  Tests  and  Autopsies 112 

Intradermal  Tests  with  Koch 's  Old  Tuberculin  and  Precipitated 
Tuberculin.     Results  and  Autopsies  113 

Typical  Cases  Selected  to  Show  that  Cattle  React  Thermally  to 
the  Intradermal  Test  119 

Showing  Tests  in  Six  Herds  in  which  Thermal  Reactions  Resulted 
from. the  Intradermal  Injection  120 

Results  of  Successive  Intradermal  Tests 121 

Comparison  of  One  Thousand  Tests  by  the  Intradermal  and  Sub- 
cutaneous Methods   124 

B,  and  C.     Results  of  Intradermal  Tests  by  European  Veterinarians.  126 


Plate  1 

Plate  2 

Plate  3 

Plate  4 

Plate  5 

Plate  6 

Plate  7 

Plate  8. 


ILLUSTRATIONS 

Figures  1  and  2,  Syringes  for  the  Intradermal  Test.     Figure  3, 
Calipers  for  Measuring  the  Reaction  145 

Figures  1  and  2,  Position  for  Injecting  Intradermally  146 

Typical  Intradermal  Reactions  147 

Measuring  Intradermal  Reactions  148 

Unusually  Large  Intradermal  Reactions  149 

Figure  1,  Swelling  caused  by  Disinfectant.     Figure  2,   Reaction 

in  Young  Calf  150 

Ophthalmic  Reactions  in  Cattle  151 

The  Intradermal  Testing  of  Swine  152 


Bulletin  243]  intradermal  test  for  tuberculosis  97 


FOREWORD 

In  all  ages,  trees  and  grasses  have  been  the  most  important  products 
of  the  soil.  The  efficient,  economic  utilization  of  grass  and  similar 
forage,  which  can  be  accomplished  only  by  the  use  of  domestic  animals, 
is  one  of  the  world's  great  problems.  It  is  a  basic  problem  in  Cali- 
fornia. Animals  are  necessary  to  utilize  the  grass  on  its  vast  ranges, 
including  valleys,  foothills,  and  mountains,  and  also  to  utilize  one  of 
the  most  important  products  of  irrigation — alfalfa.  The  production 
of  butter  fat  is  almost  an  economic  necessity  wherever  alfalfa  is 
extensively  raised.  Domestic  animals,  however,  do  more  than  convert 
into  edible  form  the  products  of  the  soil.  They  help  to  maintain  its 
fertility :  first,  by  returning  to  it  essential  fertilizing  ingredients  and, 
second,  by  permitting  a  more  diversified  system  of  agriculture. 

Obviously  one  of  the  most  important  factors  in  the  efficiency  of 
this  living  machine  is  its  health.  If  animals  when  grown  are  unfit 
for  food,  in  so  far  as  this  occurs  it  becomes  a  tax  upon  every  pound 
of  wholesome  beef.  If  the  average  usefulness  of  a  cow  extends  only 
through  three  periods  of  lactation,  as  is  claimed  to  be  the  fact  in 
some  milch  dairies,  the  cost  of  producing  every  pound  of  butter  fat 
and  every  bottle  of  milk  is  thereby  increased. 

The  most  disastrous  disease  among  cattle  is  tuberculosis.  It  is 
a  menace  that  no  stockman  or  dairyman  can  afford  to  ignore.  Its 
control  is  a  problem  that  every  state  must  seek  to  solve  as  surely  as  it 
must  endeavor  to  develop  an  abundant  food  supply. 

Of  the  eight  thousand  head  of  cattle  examined  under  the  direction 
of  the  veterinary  division  of  this  Station,  approximately  twenty-five 
hundred  have  reacted  to  the  tuberculin  test.  The  loss  from  this  disease 
in  California  alone  is  estimated  at  over  half  a  million  dollars  per  year. 
The  loss  appears  to  be  increasing.  Reports  of  meat  inspectors  in  San 
Francisco  and  Los  Angeles  indicate  that  the  proportion  of  cattle  from 
certain  ranges,  which  are  affected  with  tuberculosis,  has  increased 
from  1  to  5  per  cent  in  five  years. 

The  Agricultural  Experiment  Station  has  resolutely  set  itself  to 
study  this  problem  with  all  the  resources  at  its  command.  It  is  going 
to  be  a  long,  hard  road  to  travel.  A  thorough  study  of  the  conditions 
under  which  this  insidious  disease  is  spread  among  domestic  animals 
must  be  made.  Then  a  sane,  rational,  comprehensive  system  of  public 
control  must  be  organized.  All  evidence  points  to  the  fact  that  it 
must  be  on  lines  not  heretofore  generally  attempted  in  this  country. 


98  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

Up  to  the  present  time  the  most  efficient  weapon  with  which  to 
combat  tuberculosis  in  cattle  has  been  the  tuberculin  test.  A  descrip- 
tion of  the  usual  method  of  performing  this  test  was  published  by 
this  Station  in  Bulletin  No.  199,  entitled  "Bovine  Tuberculosis," 
which  may  be  obtained  free  upon  application.  This  method,  however, 
of  performing  the  tuberculin  test  by  injecting  the  tuberculin  sub- 
cutaneously  has  not  proved  effective  in  all  cases.  It  cannot  be  applied 
satisfactorily  to  young  calves  or  to  wild  range  cattle,  while  during 
the  hot  season  in  some  of  the  interior  valleys  the  test  has  been  unsatis- 
factory even  when  applied  to  docile  dairy  cows.  A  recent  modification 
of  the  tuberculin  test,  called  the  intradermal  method,  is  especially 
adapted  to  these  conditions.  This  bulletin  deals  with  the  best  technic 
and  the  proper  strength  of  the  tuberculin  to  be  used  in  this  new 
method,  as  well  as  its  accuracy  as  compared  with  the  usual  sub- 
cutaneous method.  The  intradermal  method  is  believed  to  constitute 
an  important  step  in  the  control  of  tuberculosis  in  both  cattle  and  hogs. 
However,  it  should  be  clearly  understood  that  a  method  of  discovering 
the  disease,  which  any  tuberculin  test  is,  is  not,  in  itself,  a  method  of 
controlling  it.  When  the  proper  knowledge  has  been  obtained  of 
the  conditions  under  which  the  disease  spreads,  a  system  of  husbandry 
must  be  developed  which  will  ensure  the  maintenance  of  healthy 
animals. 

Thomas  F.  Hunt, 

Director. 


GENERAL  CONCLUSIONS  REGARDING  THE  INTRADERMAL 
METHOD  OF  TUBERCULIN  TESTING  CATTLE 

The  intradermal  method  of  tuberculin  testing  cattle  as  first  applied 
by  the  French  investigators  Moussu  and  Mantoux37  and  as  used  under 
California  conditions  by  Ward,50  Baker,  Longley23  and  others  has  many 
advantages  over  the  usual  method.  Since  the  reaction  to  tuberculin 
when  injected  intradermally  depends  upon  a  swelling  at  the  point 
of  injection  and  not  on  a  rise  of  temperature,  it  is  to  be  preferred 
to  the  subcutaneous  under  all  conditions  that  are  liable  to  modify  the 
tuberculin  temperature  curve,  such  as  unusual  surroundings,  very  hot 
weather,  or  fatigue  from  a  long  journey.  It  can  be  satisfactorily 
applied  to  young  calves  and  wild  range  cattle  and  is  especially  useful 
in  testing  cattle  during  the  hot  season  in  the  interior  valleys  of  this 


BULLETIN  243]  INTRADERMAL  TEST  FOR  TUBERCULOSIS  99 

state  under  conditions  that  render  the  subcutaneous  method  of  testing 
unsatisfactory.  It  excels  the  subcutaneous  method  in  economy  of  time, 
labor  and  materials. 

From  our  observations  in  applying  4926  intradermal  tests,  includ- 
ing retests,  to  4001  head  of  cattle,  1614  of  which  reacted,  the  results 
being  checked  by  1000  subcutaneous  tests  and  341  autopsies  we  con- 
clude that  under  average  dairy  conditions  the  intradermal  equals  the 
subcutaneous  method  in  accuracy,  provided  a  5  per  cent,  or  stronger, 
solution  of  alcoholic  precipitated  tuberculin  is  used  and  the  test  is 
performed  by  an  experienced  operator.  We  do  not  advocate  the 
substitution  of  the  intradermal  for  the  subcutaneous  method  by  a 
veterinarian  until  he  has  become  skilled  in  its  use  by  practice  and 
observation.  The  practitioner  can  easily  accomplish  this  in  the  routine 
of  his  work  by  applying  the  two  tests  simultaneously.  Such  a  pro- 
cedure cannot  injure  the  accepted  subcutaneous  method  in  any  way, 
although  it  may  modify  the  local  intradermal  swelling  to  some  extent. 

Tuberculous  cattle  usually  react  thermally,  as  well  as  locally  to 
intradermal  injections,  even  when  very  small  doses  of  tuberculin  (1  to 
10  miligrams)  are  used.  If  temperatures  are  taken  at  two-hour 
intervals  from  the  eighth  to  the  twentieth  hours  following  the  intra- 
dermal injection  it  will  be  found  that  most  of  the  cattle  which  react 
locally  will  also  react  thermally. 

Since  neither  the  intradermal,  the  subcutaneous,  nor  the  ophthalmic 
method  will  detect  every  case  of  tuberculosis  when  applied  independ- 
ently, the  maximum  number  of  reactors  can  only  be  detected  by 
applying  the  combined  tests.  This  may  be  done  by  applying  the 
intradermal  and  subcutaneous  tests  simultaneously  or  by  administer- 
ing the  intradermal  test  alone  and  retesting  all  of  the  non-reactors 
after  an  interval  of  at  least  seven  days.  The  ophthalmic  test  may 
also  be  advantageously  combined  with  either  or  both  of  these  tests. 
The  ophthalmic  test  alone  is  unsuitable  for  use  under  the  average 
California  dairy  conditions,  except  when  only  a  few  animals  are  to 
be  tested  and  they  can  be  kept  confined  under  shelter.  For  official 
tuberculin  testing  where  a  high  degree  of  efficiency  is  necessary  a 
combination  of  the  three  tests  would  be  desirable.  The  intradermal 
method  is  especially  adapted  to  the  testing  of  swine. 


100  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


RESULTS  OBTAINED  WITH  TUBERCULIN  IN  TESTING 

CATTLE  AND  HOGS  BY  THE  INTRADERMAL 

AND  OTHER  METHODS 


BY 

CLARENCE  M.  HAEING 


The  intradermal  or  intracutaneous*  method  of  applying  the 
tuberculin  test  to  cattle  is  now  regularly  used  by  several  veterinary 
practitioners  in  California,  and  reports  from  other  states  and  from 
foreign  countries  indicate  that  the  method  is  rapidly  gaming  favor 
among  veterinarians  elsewhere.  The  test  possesses  such  advantages 
in  economy  of  time  and  materials  that  it  has  been  widely  accepted  by 
practitioners  as  a  substitute  for  the  subcutaneous  method,  without 
waiting  to  determine  whether  it  is  sufficiently  accurate  to  warrant 
such  an  acceptance.  The  data  concerning  its  accuracy,  as  compared 
with  that  of  the  usual  subcutaneous  method,  are  not  sufficient,  however, 
to  convince  many  state  officials  of  the  advisability  of  accepting  the 
intradermal  test  as  a  substitute.  Another  new  method  of  tuberculin 
testing,  called  the  ophthalmic,  conjunctival  or  eye  test,  is  used  in 
some  sections.  The  ophthalmic  method,  although  not  popular  in  this 
country,  is  preferred  by  some  veterinarians.  In  regard  to  the  accept- 
ance of  these  methods  for  the  admission  of  cattle  into  states  requir- 
ing a  tuberculin  test  certificate,  the  following  information  is  of 
interest.  Statements  have  been  received  by  the  writers  from  the  live- 
stock sanitary  authorities  of  thirty-nine  states.  In  Delaware  and  Cali- 
fornia the  intradermal  test  is  accepted,  if  performed  by  an  approved 
veterinarian.  The  State  Veterinarian  of  Pennsylvania  writes  that  he 
would  not  recommend  the  livestock  sanitary  board  to  recognize  the 
method  indiscriminately  except  under  certain  conditions  when  the 
t  intradermal  or  ophthalmic  tests  were  made  by  an  unusually  well- 
qualified  veterinarian  and  a  certificate  for  physical  examination 
accompanied  a  report  of  the  test.  The  State  Veterinarian  of  Missouri 
writes:  "We  are  using  the  intradermal  test  exclusively  in  our  state 


*  The  terms  intradermal  and  intracutaneous  are  synonyms.  Custom  renders 
the  word  intradermal  preferable,  when  mentioned  in  connection  with  the  testing 
of  cattle. 


Bulletin  243]  intradermal  test  for  tuberculosis  101 

work,  but  for  interstate  shipments  we  are  using  the  thermal  method, 
for  the  reason  that  the  intradermal  leaves  no  record.  Therefore, 
until  such  time  as  an  agreement  can  be  made  between  the  different 
states  and  there  is  a  better  system  for  the  control  of  the  veterinarians, 
it  will  be  necessary  for  us  to  require  the  thermal  test  and  a  record 
of  the  same  filed  in  this  office  immediately  after  the  inspection  is 
made.  "We  have  the  greatest  confidence  in  the  intradermal  test  and 
would  not  hesitate  to  accept  shipments  where  it  has  been  applied, 
except  for  the  fact  that  only  a  very  small  per  cent  of  the  veterinarians 
doing  interstate  work  are  familiar  with  the  intradermal  test.  There- 
fore, I  see  no  possibility  of  our  accepting  the  intradermal  test  in  the 
near  future."  Most  of  the  state  authorities  agree  that  they  cannot 
as  yet  officially  accept  either  the  intradermal  or  ophthalmic  method. 
The  concensus  of  opinion  seems  to  be  that  these  tests  are  fairly  satis- 
factory when  administered  by  careful  and  experienced  operators,  but 
that  it  is  unsafe  to  accept  such  tests  from  veterinarians  indiscrim- 
inately, until  these  methods  have  come  into  more  general  use  and  have 
been  accepted  by  the  United  States  Bureau  of  Animal  Industry. 


LOCAL    TUBERCULIN   REACTIONS   AND    THEIR   USE    FOR   DIAGNOSIS 
IN  HUMAN  AND  VETERINARY  PRACTICE 

In  human  practice*  the  intradermal  method  is  used  to  some  extent, 
but  many  physicians  consider  this  method  too  delicate  for  routine 
diagnosis  in  man.  Their  objection  is  that  the  test  is  so  searching  in 
its  scrutiny  that  it  causes  reactions  in  latent  cases  which  would 
probably  never  develop  active  tuberculosis. 

In  veterinary  practice,  however,  this  "searching  scrutiny"  is 
usually  desired  and  from  data  already  available  there  is  reason  to 
believe  that  this  test  when  combined  with  observations  concerning  the 


*  The  tissues  of  tuberculous  persons  and  animals  are  sensitive  to  tuberculin. 
This  sensitiveness  is  most  marked  in  the  tissues  that  are  actually  invaded  by 
the  germs,  but  it  also  exists  to  some  extent  in  the  other  tissues  of  the  body 
which  contain  blood  vessels.  The  living  tissues  of  individuals  who  have  not 
been  infected  with  tuberculosis  show  no  sensitiveness  to  tuberculin. 

Attention  was  first  called  to  the  possibilities  of  the  intradermal  injection 
of  tuberculin  as  a  means  of  diagnosis  by  Mendel,  who  suggested  that  the  pro- 
cedure followed  in  the  intradermal  method  of  injecting  for  anesthesia  would  be 
a  delicate  and  satisfactory  means  of  diagnosis.  The  suggestion  was  soon  put 
into  practice  by  Mantoux  and  others.  According  to  Hamman  and  Wolman,12 
the  intradermal  method  when  applied  to  man  has  no  equal  as  a  means  of 
estimating  the  degree  of  tuberculin  hyper-sensitiveness.  They  conclude  from 
a  study  of  the  records  of  a  large  number  of  tests  made  by  the  various  methods 
of  tuberculin  testing  that  the  intradermal  and  subcutaneous  local  tests  are  the 
most  delicate  tests  for  man.  They  reveal  practically  the  full  percentage  of 
tuberculosis  infected  individuals.     In  the  order  of  their  sensitiveness  the  tests 


102  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

thermal  reaction  furnishes  a  more  accurate  means  of  diagnosis  than 
the  usual  subcutaneous  method  of  testing  cattle,  in  which  no  atten- 
tion is  paid  to  the  local  reaction  and  no  attempt  is  made  to  select 
and  prepare  the  field  of  injection  and  to  inject  in  such  a  way  that 
if  the  local  reaction  occurs  it  will  be  easily  detected.  Many  veterin- 
arians in  making  the  subcutaneous  test  shave  the  neck  at  the  point 
where  the  subcutaneous  injection  is  to  be  made,  in  order  to  more 
easily  observe  the  local  reaction  which  frequently  occurs.  Corbett 
and  Griffiths6  state  that  the  local  reactions  resulting  from  subcutaneous 
injections  in  the  neck  were  usually  very  slight  and  of  no  special  value. 
However,  we  have  found  that  in  interpreting  doubtful  thermal  re- 
actions it  is  of  assistance  to  examine  the  site  of  injection  for  local 
swellings.  One  of  us  (Bell)  has  found  it  of  value,  in  testing  by  the 
subcutaneous  method,  to  inject  the  tuberculin  through  the  skin  of 
the  hindquarter  over  the  semimembranosis  muscle.  He  finds  that  the 
local  reactions  to  the  subcutaneous  injections  are  more  clearly  defined 
than  in  the  loose  tissues  of  the  neck.  The  intradermal  method  excels 
the  subcutaneous  in  the  size  and  defmiteness  of  its  local  reactions. 


TECHNIC  OF  THE   INTRADERMAL   METHOD 

The  manner  in  which  the  intradermal  differs  from  the  usual  sub- 
cutaneous test  is  that  the  tuberculin  is  injected  in  y20  to  14  cc. 
amounts  into  the  deeper  layers  of  the  skin,  or  into  the  subdermal 
tissue  immediately  adjacent,  instead  of  injecting  a  large  quantity 
(1  to  4  cc.)  deeply  under  the  skin.  The  most  suitable  place  to  make 
this  injection  is  in  one  of  the  folds  of  the  skin  on  the  under  side  of 
the  base  of  the  tail.  We  have  used  the  skin  of  the  neck  in  some  of 
our  tests,  but  find  the  subcaudal  fold  more   convenient.      The  skin 


for  tuberculosis  in  man  arrange  themselves  according  to  Hamman  and  Wolman11 
as  follows: 

Intracutaneous  test   (Intradermal) 

Subcutaneous-local  test   (Strichreaction) 

Cutaneous  test   (von  Pirquet) 

Subcutaneous  test   (Koch) 

Percutaneous  test   (Moro  inunction) 

Conjunctival  test  (Calmette  ophthalmic). 
As  a  means  of  diagnosis  of  tuberculosis  in  man,  the  first  two  tests  are  said  by 
these  writers  to  be  undesirable  for  the  very  reason  that  they  detect  even  the 
latent  cases  of  infection.  They  state  that  in  adult  life  and  even  in  late  child- 
hood the  subcutaneous  local  reaction  is  of  absolutely  no  diagnostic  value,  since 
only  an  occasional  individual  will  escape  its  searching  scrutiny.  What  little 
diagnostic  value  it  has  is  restricted  to  the  first  two  years  of  life  and  even  at 
that  age  it  has  no  marked  value  above  the  cutaneous  test  for  the  reason  that 
these  tests  have  shown  the  astonishing  extent  to  which  man  is  tuberculosis- 
infected  and  at  what  an  early  age  the  infection  occurs. 


Bulletin  243]  intradermal  TEST  FOR  TUBERCULOSIS  103 

here  is  free  from  hair  and  is  soft  and  pliable.  A  hypodermic  syringe 
with  a  short  needle  point  should  be  used.  Such  a  syringe  should 
have  a  25  or  26  gage  needle  with  a  point  3/16  to  14  of  an  inch  in 
length,  similar  to  those  used  by  dentists  for  injecting  local  anaesthetics 
(see  plate  1,  figures  1  and  2). 

The  subcaudal  fold  is  grasped  between  the  thumb  and  the  first 
two  fingers  of  the  left  hand  and  the  needle  inserted  horizontally  into 
the  thickness  of  the  skin  grasped  between  the  thumb  and  finger  (see 
plate  2,  figure  1).  The  y10  or  %  cc.  dose,  if  properly  placed,  can  be 
felt  in  the  layers  of  the  skin  as  it  is  expelled  from  the  syringe,  where 
it  remains  as  a  small  lump  in  the  skin  after  the  needle  has  been 
removed.  In  our  first  tests  we  made  the  mistake  of  trying  to  inject 
as  near  the  surface  of  the  skin  as  possible.  It  is  difficult  to  inject 
into  the  layers  of  the  epidermis  and  an  injection  into  this  part  of 
the  skin  is  of  little  diagnostic  value.  In  case  the  needle  is  of  the 
proper  length,  namely,  one-quarter  of  an  inch,  there  is  little  danger 
of  going  completely  through  the  skin.  When  the  proper  point  in 
the  subcaudal  fold  is  selected,  it  makes  little  difference  whether  the 
point  of  the  needle  is  in  the  derma  or  in  the  subdermal  connective 
tissue.  With  the  proper  syringe  an  expert  operator  can  inject  in 
the  dark  as  accurately  as  in  a  good  light.  We  have  found  that 
characteristic  reactions  occur  with  the  injections  from  a  long  needle, 
placed  completely  through  and  beneath  the  skin  layers  of  the  sub- 
caudal fold.  It  is  well  to  inject  at  a  point  on  the  fold  about  two 
and  one-half  or  three  inches  down  the  tail  from  the  anus,  since 
elsewhere  reactions  are  not  so  easily  perceived  and  at  this  point  the 
bone  and  solid  tissue  of  the  tail  form  a  background  which  renders 
the  local  reactions  more  prominent  than  those  of  the  skin  of  the  neck. 

After  successfully  using  the  intradermal  method  on  fifteen  hundred 
head  of  cattle,  Longley23  has  expressed  the  opinion  that  it  is  impos- 
sible to  inject  into  the  skin  proper,  but  that  the  dose  goes  into  the 
subdermal  connective  tissue.  From  experiments  which  we  have  made 
on  the  hides  of  recently  killed  cattle,  we  think  he  must  have  meant 
the  epidermis.  When  a  one-quarter  inch  needle  is  inserted  slightly 
horizontally  into  the  subcaudal  fold  it  rarely  penetrates  deeper  than 
the  lower  layers  of  the  derma  or  true  skin. 

In  injecting  cattle  that  are  secured  in  stanchions,  a  convenient 
way  is  to  stand  by  the  side  of  the  cow,  as  shown  in  plate  2,  figure  1, 
pressing  the  knee  into  the  animal's  flank  and  grasping  the  loose  folds 
of  skin  from  under  the  tail  without  raising  the  tail.  Some  operators, 
when  injecting,  prefer  to  stand  directly  behind  the  cow,  as  shown  in 
plate  2.  figure  2,  but  this  method  is  impracticable  except  with  very 


104  UNIVERSITY  OF  CALIFORNIA — EXPERIMENT  STATION 

docile  cattle  or  when  an  assistant  is  available  to  steady  the  animal 
and  keep  her  from  moving  from  side  to  side. 

We  deem  it  impracticable  to  disinfect  or  otherwise  prepare  the 
subcaudal  fold  for  the  intradermal  injection,  except  when  it  is  soiled. 
In  such  cases,  wipe  the  fold  clean  with  a  rag,  apply  50  per  cent  alcohol 
with  absorbent  cotton  and  wipe  with  dry  cotton.  Before  injecting 
each  animal,  the  syringe  needle  should  be  dipped  in  strong  disin- 
fectant, and  wiped  dry  with  sterile  absorbent  cotton.  After  dipping 
the  needle  in  an  irritating  disinfectant,  take  pains  to  wipe  it  off  before 
inserting  it,  for  the  disinfectant  may  cause  irritation  and  produce  a 
swelling  which  might  be  mistaken  for  a  reaction.  The  application  of 
strong  disinfectants  at  the  point  of  intradermal  injection  should  be 
avoided.  In  several  instances  we  have  observed  swellings,  varying  in 
size  from  a  hazelnut  to  a  hen's  egg,  on  the  subcaudal  fold  as  a  result 
of  the  application  of  strong  lysol  solution.  These  might  have  been 
mistaken  for  reactions,  but  for  the  fact  that  the  cattle  were  not 
injected  with  tuberculin  (see  plate  6,  figure  1). 

KIND  AND  STEENGTH  OF  TUBERCULIN  FOR  INTRADERMAL  USE 

From  experiments  which  we  have  conducted  during  the  past  year, 
we  believe  that  a  strong  solution  of  alcoholic  precipitated  tuberculin* 
is  most  suitable  for  use  with  the  intradermal  test  in  cattle.     In  order 


*  Laboratories  vary  considerably  in  their  methods  of  preparing  precipitated 
tuberculin.  Most  of  the  tuberculin  which  we  have  used  has  been  prepared  as 
follows:  To  twenty  volumes  of  absolute  alcohol  add  very  slowly  one  volame  of 
Koch's  Old  Tuberculin  concentrated.  Stir  thoroughly  and  set  aside  for  twenty- 
four  hours.  At  the  end  of  this  time  a  floculent  deposit  will  be  seen  in  the  bottom 
of  the  vessel.  The  supernatant  fluid  is  decanted  and  the  partially  dried  pre- 
cipitate redissolved  in  one  volume  of  1  per  cent  sodium  chloride  solution  and  the 
tuberculin  reprecipitated  by  adding  this  solution  to  twenty  volumes  of  absolute 
alcohol.  The  supernatant  fluid  is  decanted  and  the  precipitate  washed  by  adding 
it  to  nine  volumes  of  absolute  alcohol.  The  precipitate  is  then  placed  on  clay 
plates,  put  in  a  dessicator  and,  under  vacuum,  dried  over  sulphuric  acid.  A 
brownish  powder  can  be  scraped  from  the  plate.  This  is  the  precipitated  tuber- 
culin; also  known  as  dry  tuberculin  or  tuberculin  siccum.  This  tuberculin  is 
said  to  be  soluble  in  the  proportion  of  one  part  by  weight  to  ten  of  water,  but 
higher  concentrations  can  sometimes  be  obtained.  We  have  been  able  to  make 
50  per  cent  solutions.  According  to  Brieger  and  Proskauer,8  one-tenth  of  a  gram 
of  this  precipitated  tuberculin  produces  the  same  effect  as  five-tenths  of  a  gram 
of  crude  tuberculin  (Koch's  Old).  According  to  Hamman  and  Wolman  this 
kind  of  tuberculin  seems  to  be  more  toxic  to  animals  than  Koch's  Old  Tuberculin. 

This  may  be  dispensed  in  powder  form  to  be  made  into  a  solution  when  needed. 
Most  laboratories,  however,  prepare  the  solution  before  shipment,  a  diluent 
of  %  per  cent  of  trikresol  being  added  and  the  solution  sterilized  by  steaming 
at  60°  C.  for  thirty  minutes. 

The  concentrated  tuberculin  used  in  preparing  the  precipitated  should  be 
made  from  selected  cultures.  The  culture  media  used  should  be  prepared  accord- 
ing to  standard  methods,  using  veal  broth  and  not  that  prepared  from  com- 
mercial extract  of  beef.  We  have  found  some  of  the  precipitated  tuberculin 
on  the  market  to  be  of  low  potency. 


Bulletin  243]  intradermal  test  for  tuberculosis  105 

to  obtain  a  distinct  and  well-marked  subcaudal  reaction  this  purified 
tuberculin  should  be  used  in  at  least  a  5  per  cent  solution  (50  milli- 
grams per  cc.  of  physiological  salt  solution)  administering  doses  of 
between  y10  and  %  cc.  The  injection  of  ten  times  this  dose  (%  cc. 
of  25  per  cent  solution)  subcaudally  leaves  no  perceptible  swelling  in 
non- tuberculous  animals  after  the  forty-eighth  hour  while  in  tuber- 
culous cattle  a  swelling  the  size  of  an  egg  usually  results.  Vallee48 
and  his  co-workers  endeavored  to  obtain  the  subcaudal  reaction  with 
the  minimum  dose,  but  in  order  to  obtain  the  maximum  number  of 
reactions  in  tuberculous  cattle,  we  believe  it  is  desirable  to  use  a 
stronger  solution  than  they  suggest.  This  point  is  discussed  later  in 
connection  with  tuberculin  manufactured  by  various  firms.  Ten  per 
cent  solutions  of  potent  precipitated  tuberculin  (100  milligrams  per 
cc.  of  physiological  salt  solution)  will  usually  produce  pronounced 
local  and  thermal  reactions  in  tuberculous  cattle,  when  administered 
intradermally. 

Koch's  Old  Tuberculin  in  10  per  cent  solution  has  also  given  good 
results  as  can  be  seen  by  an  inspection  of  Tables  1  and  2.  The  tuber- 
culin distributed  by  the  Bureau  of  Animal  Industry  for  official  use 
is  equivalent  to  10  per  cent  Koch's  Old  Tuberculin  and  for  the 
practictioner  who  can  obtain  it  this  tuberculin  will  doubtless  prove 
more  satisfactory  than  the  solutions  of  tuberculin  sold  for  intradermal 
use  by  biological  houses. 

We  have  also  obtained  excellent  results  in  both  the  intradermal  and 
ophthalmic  work  with  phymatin,  a  tuberculin  of  unknown  content 
manufactured  in  Germany. 

Hutyra  and  Marek15  have  suggested  that  the  tuberculins  which 
contain  ground  or  pulverized  tubercle  bacteria  will  probably  be  found 
to  be  most  potent  in  producing  local  reactions.  In  view  of  this  we 
tried  two  such  preparations  which,  by  courtesy  of  the  Cutter  Labora- 
tory, were  manufactured  especially  for  these  experiments.  One  of 
these  consisted  of  a  glycerin  free  suspension  of  finely  ground  tubercle 
bacilli.  The  other  consisted  of  a  similar  suspension  of  whole  tubercle 
bacilli  killed  by  steaming  for  two  hours  at  60°  C.  The  emulsions 
contained  a  solid  content  of  from  10  to  20  milligrams  tubercle  bacilli 
per  cc.  In  tuberculous  animals  no  local  or  thermal  reactions  resulted 
from  the  intradermal  injection  of  Y10  cc.  doses  of  these  suspensions. 

Precautions  Against  the  Deterioration  of  Precipitated  Tuberculin. 
— Meyer33  has  called  attention  to  the  fact  that  aqueous  solutions  of 
alcoholic  precipitated  mallein  disintegrate  rapidly,  due  in  part  to  the 
absence  of  preservative  and  in  part  to  changes  in  toxin  molecules. 
Therefore,  the  powder  form  alone  can  be  kept  in  stock  and  the  test 


106  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

solution  should  always  be  made  shortly  before  use.  It  is  probable 
that  this  holds  true  for  solutions  of  precipitated  tuberculin  as  well. 
As  a  diluent  for  the  dry  tuberculin  we  have  used  14  per  cent  solutions 
of  trikresol  in  physiological  salt  solution  and,  if  the  dilution  is  pre- 
pared aseptically,  we  have  found  that  the  tuberculin  dilution  will 
remain  sterile  and  potent  for  months.  The  use  of  trikresol  or  phenol 
in  %  to  y2  per  cent  solutions  for  the  intradermal  test  does  not 
appear  to  produce  a  perceptible  local  irritation  or  be  objectionable  in 
any  way.  On  account  of  the  possibility  of  molecular  disintegration, 
however,  we  advise  that  the  test  solution  be  prepared  shortly  before 
use.  It  is  convenient  to  keep  on  hand  a  stock  of  sterile  2  cc.  amber 
glass  bottles,  each  bottle  containing  one-tenth  of  a  gram  of  dry  alcoholic 
precipitated  tuberculin.  By  adding  to  one  of  these  2  cc.  of  sterile 
physiological  salt  solution,  ten  doses  of  5  per  cent  tuberculin  solution 
can  be  prepared.  As  the  tuberculin  powder  goes  into  solution  very 
slowly,  the  diluent  should  be  added  at  least  two  hours  before  the 
injection  is  to  be  made.  On  account  of  the  detrimental  effect  of  light 
on  such  solutions  they  should  be  kept  in  amber  bottles  and  protected 
from  direct  sunlight  as  much  as  possible. 

Lack  of  Uniformity  in  Tuberculin. — In  the  United  States  there  is 
a  great  lack  of  uniformity  regarding  the  tuberculin  used.  We  have 
purchased  "intradermal"  tuberculin  from  six  different  firms,  and  in 
only  two  instances  has  the  product  been  the  same,  as  is  shown  by  the 
following : 

Firm  A  recommends  for  the  intradermal  test  in  cattle  a  tuberculin  bearing 
the  label  "Tuberculin  O.  T.  (Original)  Serial  Dilution  No.  5,  lcc.  contains 
100  mg."  This  they  state  is  prepared  by  diluting  one  part  of  Koch's  Old 
Tuberculin  with  nine  parts  of  physiological  salt  solution  containing  %  per  cent 
trikresol. 

Recently  this  firm  has  placed  on  the  market  "Intradermal  Tuberculin, 
Veterinary, ' '  which  is  essentially  the  same  as  the  above  in  strength,  although 
in  its  preparation  it  is  not  deemed  necessary  to  concentrate  the  original  culture 
down  to  the  full  equivalent  of  Koch 's  old  tuberculin  before  diluting  it  back 
to  the  strength  of  dilution  No.  5. 

On  request  they  will  also  furnish  for  the  intradermal  test  dilutions  of 
alcoholic  precipitated  tuberculin,  glycerin  free,  in  any  dilution  desired  up  to 
10  per  cent  solution. 

Firm  B  recommends  for  the  intradermal  test  of  cattle  a  tuberculin  bearing 
the  label  "Solution  Purified  Tuberculin  for  Intradermal  Test.  Each  cc.  contains 
50  milligrams  purified  and  precipitated  Tuberculin  in  sterilized  normal  salt 
solution.  Dose — 1  to  5  minims."  On  request,  this  firm  will  also  furnish  for  the 
intradermal  test  stronger  solutions  of  alcoholic  precipitated  tuberculin. 


Bulletin  243]  intradermal  test  for  tuberculosis  107 

Firm  C  recommends  for  the  intradermal  test  in  cattle  "Ordinary  Veterinary 
Tuberculin."  This  is  presumably  similar  to  that  distributed  by  the  Bureau 
of  Animal  Industry  for  the  use  of  state  and  municipal  officials. 

Firm  D  states  that  they  do  not  dispense  any  especially  prepared  tuberculin 
for  the  intradermal  test,  but  recommends  their  "concentrated  tuberculin." 
The  label  on  this  tuberculin  reads  as  follows:  "Tuberculin,  Veterinary,  ready 
for  use."  This  firm  advises  us  by  letter  that  this  concentrated  tuberculin  is 
seven  times  the  strength  of  the  ordinary  culture  and  contains  35  per  cent 
glycerin. 

Finn  E  recommends  a  50  per  cent  solution  of  Koch's  old  tuberculin.  This 
corresponds  with  the  recommendations  of  Hutyra  and  Marek,  Joseph  and  others. 
This  firm  writes  that  they  also  dispense  a  purified  tuberculin  of  strength 
equivalent  to  the  above  for  intradermal  use.  They  are  not  able  to  state  the 
method  of  the  preparation  of  this  tuberculin,  as  it  is  manufactured  in  a  foreign 
laboratory. 

Firm  F  recommends  tuberculin  especially  prepared  for  the  intradermal  test- 
ing of  cattle.  This  firm  could  not  give  any  particulars  regarding  the  contents 
of  the  tuberculin  or  its  method  of  preparation,  as  it  was  manufactured  in  a 
foreign  laboratory. 


In  view  of  the  diversity  of  tuberculins  recommended  for  the  intra- 
dermal test,  the  following  information  concerning  the  relative  merits 
of  alcoholic  precipitated  tuberculin  versus  Koch's  old  tuberculin  is 
of  interest : 

Alcoholic  Precipitated  Tuberculin. — Using  alcoholic  precipitated 
tuberculin  in  solutions  varying  from  y2  to  50  per  cent,  we  have  tested 
by  the  intradermal  method  2312  cattle,  1190  of  which  reacted  with 
clear  and  positive  results.  In  one  instance  where  dry  alcoholic  pre- 
cipitated tuberculin  was  purchased  from  a  certain  laboratory  and  used 
intradermally  on  tuberculous  cattle  in  10  per  cent  solutions  it  gave 
very  weak  or  negative  results.  Tests  made  with  "purified  tuberculin" 
of  foreign  manufacture  and  unknown  strength  have  also  given  unsatis- 
factory results.  We  are  now  making  tests  on  guinea-pigs  to  show  the 
comparative  toxicity  of  tuberculins  from  various  manufacturers,  the 
preliminary  results  of  which  indicate  that  tuberculins  sold  in  this 
country  vary  considerably  in  strength.  Government  supervision  and 
standardization  of  biological  products,  especially  tuberculin,  is  greatly 
needed.  When  a  potent  precipitated  tuberculin  is  not  available  it  is 
preferable  to  Koch's  Old  Tuberculin  for  intradermal  use.  Our  reasons 
for  prefering  the  precipitated  tuberculin  to  dilutions  of  Koch's  Old 
Tuberculin  are  as  follows : 


108  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

In  thirty-two  of  our  intradermal  tests  with  Koch's  Old  Tuberculin, 
the  results  were  checked  by  injecting  the  opposite  subcaudal  fold  with 
y5  cc.  of  physiological  salt  solution,  containing  5  per  cent  glycerin 
and  y2  per  cent  phenol,  a  new  clean  syringe  being  used.  The  swellings 
produced  by  the  glycerin  in  two  cases  were  persistent  enough  to  be 
confusing.  We  then  undertook  extensive  trials  concerning  the  effect 
of  solutions  of  glycerin,  phenol  and  trikresol  when  injected  intra- 
dermal^ and  found  that  the  glycerin  in  solutions  of  5  per  cent  or 
stronger  would  frequently  produce  swellings  large  enough  to  be  con- 
fusing when  injected  in  Vs  cc-  doses.  The  trikresol  and  phenol  in 
3/2  per  cent  solutions  on  intradermal  injection  did  not  produce 
swellings. 

We  also  tried  the  effect  of  injecting  10  per  cent  solutions  of  the 
precipitates  derived  by  treating  concentrated  glycerin  bouillon  with 
alcohol  in  a  manner  similar  to  that  used  in  producing  precipitated 
tuberculin.  These  did  not  produce  swellings  large  enough  to  be 
confused  with  an  intradermal  reaction. 

Our  observations  in  this  respect  corroborate  the  observations  of 
Vallee,  Declaire  and  Herbert.48  They  state  that  many  healthy  cattle, 
subjected  to  the  intradermal  test  according  to  the  method  of  Moussu 
and  Mantoux,35-37  show  in  about  6  per  cent  of  the  cases  an  immediate 
local  swelling,  at  periods  of  time  varying  from  five  to  sixty  minutes 
after  injection.  This  swelling  at  the  site  of  the  injection  is  edematous 
in  character  and  varies  in  size  from  the  dimensions  of  a  hazelnut  to 
those  of  a  plum.  The  swelling  disappears  quickly,  rarely  persisting 
more  than  twenty-four  hours,  differing  in  this  respect  from  the  specific 
reaction  in  tuberculous  animals,  which  is  usually  more  persistent. 
Some  cases,  however,  .in  healthy  cattle  persist  more  than  forty-eight 
hours.  The  above  authorities  assume  that  the  swelling  is  brought  about 
in  part  by  the  glycerin  or  by  certain  salts  in  the  bouillon  and  that  it 
may  be  in  part  a  reaction  to  a  traumatism.  According  to  Mulford's 
Veterinary  Bulletin,  they  recommend  the  use  of  y>  per  cent  pre- 
cipitated tuberculin  solution  with  %  cc.  doses. 

Under  certain  conditions,  when  the  intradermal  is  to  be  followed 
by  a  subcutaneous  test,  it  might  be  desirable  to  use  this  weak  solution 
of  tuberculin,  as  there  would  be  less  likelihood  of  its  having  an 
inhibitory  effect  on  the  subsequent  test.  In  fact,  Hamman  and  AVolman 
claim  there  is  reason  to  believe  that  the  injection  of  y10  cc.  doses  of 
1/2  per  cent  tuberculin  in  a  human  being  may  have  a  sensitizing  effect 
which  will  render  a  subsequent  test  more  effective.  We  have  no 
evidence  to  show  that  this  holds  true  for  cattle. 


Bulletin  243]  intradermal  TEST  FOR  TUBERCULOSIS  109 

Foth11  states  that  the  dose  used  by  the  French  authors  was  too 
small  to  get  the  maximum  number  of  local  reactions.  This  is  probably 
true,  but  from  our  experiments  we  believe  that  the  quantity  of  liquid 
injected  should  not  exceed  y5  cc.  and  that  it  is  better  to  increase  the 
concentration  of  the  tuberculin  instead  of  increasing  the  quantity  of 
the  dose.  Most  authorities  prefer  to  use  Koch's  old  tuberculin  for 
the  intradermal  test  and  we  would  be  inclined  to  agree  in  this  if  it 
were  not  for  its  glycerin  content. 

Koch's  Old  Tuberculin  (Tuberculin  0.  T.  Original). — Hutyra  and 
Marek14  state  that  a  50  per  cent  solution  of  Koch's  old  tuberculin, 
when  injected  intradermally,  produces  good  clear  results  in  both  posi- 
tive and  negative  cases.  Several  veterinarians  who  have  had  exten- 
sive experience  in  the  use  of  tuberculin  inform  me  that  the  ordinary 
B.  A.  I.  tuberculin,  undiluted,  in  doses  of  three  to  five  drops,  has  given 
excellent  results  and  that  from  a  long  series  of  tests  in  actual  practice 
they  do  not  believe  it  necessary  to  use  purified  tuberculin. 

Joseph16  has  stated  that  not  over  %  cc.  should  be  injected,  and 
we  believe  from  our  work  that  his  suggestions  should  be  followed, 
provided  a  strong  solution  of  tuberculin  is  used.  Joseph  used  y20  cc. 
of  Koch 's  old  tuberculin  diluted  with  an  equal  amount  of  physiological 
salt  solution,  which  would  make  a  solution  containing  25  per  cent 
glycerin. 

Using  Koch's  old  tuberculin  according  to  the  method  of  Moussu 
and  Mantoux,37  we  have  tested  by  the  intradermal  method  1942 
cattle,  477  of  which  reacted  with  clear  and  positive  results.  We  have 
concluded,  however,  after  due  experimentation  and  consideration  of 
the  recommendations  of  the  investigators  and  firms  above  mentioned, 
that  a  strong  solution  of  alcoholic  precipitated  tuberculin  is  preferable 
to  Koch's  old  tuberculin  for  the  intradermal  test  in  cattle. 

The  summary  given  in  Table  1  is  of  interest  in  considering  the 
relative  merits  of  the  various  kinds  and  strengths  of  tuberculin  recom- 
mended for  the  intradermal  method. 

In  addition  to  the  tests  mentioned  in  Table  1,  we  have  experimented 
with  aqueous  glycerin  free  suspensions  of  finely  ground  tubercle 
bacilli,  also  with  suspension  of  whole  tubercle  bacilli  which  had  been 
killed  by  heat,  and  with  phymatin,  a  German  preparation  of  unknown 
composition  highly  recommended  for  the  ophthalmic  and  intradermal 
tests. 

A  summary  of  the  autopsies  on  tested  cattle  is  given  in  Table  2. 


110 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


TABLE   1 

Eesults  from  the  Intradermal  Use  of  Various  Solutions  of  Tuberculin 


Kind  of  tuberculin 

Koch's  old  tuberculin 
dilution  1  to  10  of 
physiological  salt 
solution. 


Concentrated  tuberculin 
equal  in  strength  to 
a  70  per  cent  solu- 
tion of  Koch's  old 
tuberculin. 

Koch's  old  tuberculin 
undiluted. 


Alcoholic  precipitated 
tuberculin  10  per 
cent  solution  (100 
milligrams  per  cc.  of 
physiological  salt 
solution). 

Alcoholic  precipitated 
tuberculin  5  per  cent 
solution  (50  milli- 
grams per  cc). 

Alcoholic  precipitated 
tuberculin  2  per*  cent 
solution  (20  milli- 
grams per  cc). 

Alcoholic  precipitated 
tuberculin  \  per  cent 
solution  (5  milli- 
grams per  cc). 


A  tuberculin  of  foreign 
manufacture  recom- 
mended for  intra- 
dermal use.  Strength 
unknown. 


Dose 


i/10  to 
%  cc 


Total 
number  of 
cows  tested 


1942 


y5  cc 


15 


i/10  cc  24 


i/20to 

i/5  cc         201 


Vioto 

y5  cc         937 


y5  cc       1055 


y5  cc.      ii9 


y5  cc        17 


Total 

number 

of  reactions 

477 


n 


48 


630 


479 


33 


Conclusions  regarding  the 
tuberculin 

Fairly  satisfactory.  Produces 
distinct  local  reaction  swell- 
ings, but  the  glycerin  which 
it  contains  causes  a  swell- 
ing immediately  after  in- 
jection which  occasionally 
persists  long  enough  in 
healthy  cattle  to  be  con- 
fusing. Thermal  reactions 
also  occur  with  this  dosage. 

The  tuberculin  contains  35 
per  cent  glycerin.  The 
glycerin  content  is  objec- 
tionable. 

Used  on  24  head  of  non-re- 
acting cattle.  The  swell- 
ings due  to  the  glycerin  in 
some  cases  were  four  inches 
in  diameter  and  persisted 
over  48  hours. 

This  kind  of  tuberculin  in 
strengths  of  5  to  50  per 
cent  is  very  satisfactory. 
It  produces  pronounced 
local  and  thermal  reactions 
when  injected  intradermally 
in  y10  cc  doses.  No  local 
swellings  occur  in  non- 
tuberculous  cattle. 

Fairly  satisfactory.  Produces 
thermal  as  well  as  local  re- 
actions when  injected  in- 
tradermally. A  stronger 
solution  is  better. 

Too  weak  except  when  the 
cattle  are  to  be  retested 
with  stronger  tuberculin. 
This  strength  of  tuberculin 
produces  moderate  local  and 
thermal  reactions  when  in- 
jected intradermally. 

The  firm  selling  this  tuber- 
culin was  unable  to  state 
its  strength,  saying  it  was 
manufactured  in  a  foreign 
country.  The  autopsies 
proved  this  tuberculin  un- 
satisfactory. 


Total  tests. 

4310 

1682 

Subtracting    for    re- 

tests. 

521 

136 

Autopsies  were  made  in  341 
cases.     See  notes  following. 


Number  of  cattle  tested. 


1546 


Bulletin  243]  intradermal  test  for  tuberculosis  111 

Autopsy  Notes 

Herd  No.  1. — Fifteen  cattle,  which  showed  physical  signs  of  tuberculosis, 
were  injected  with  %  cc.  of  a  10  per  cent  dilution  of  Koch's  old  tuberculin,  and 
fourteen  reacted.  One  week  later  they  were  tested  by  the  subcutaneous  method 
and  all  reacted.     On  autopsy  they  were  found  to  be  tuberculous.     (See  Table  6.) 

Herd  No.  3. — In  testing  a  herd  of  209  cattle,  using  the  intradermal  and 
subcutaneous  methods  simultaneously,  one  cow  reacted  to  the  subcutaneous  test 
and  another  to  the  intradermal  test.  On  autopsy  these  two  were  found  tuber- 
culous.    (See  cases  9  and  11,  p.  132;  also  Table  6.) 

Herd  No.  6. — Two  cattle  that  reacted  locally  to  an  intradermal  injection  of 
%  cc.  of  a  10  per  cent  dilution  of  Koch's  old  tuberculin  failed  to  react  to  sub- 
cutaneous tests  applied  three  to  thirty  days  later.  They  were  found  tuberculous 
on  autopsy.     (See  cases  3  and  4,  p.  130.) 

Herd  No.  8. — One  hundred  and  seventy-two  cattle,  when  tested  after  the 
method  of  Moussu,  gave  124  reactions.  Only  two  of  these  were  autopsied,  and 
both  were  found  tuberculous.  In  addition,  we  observed  thirty  cows  in  this  herd 
that  evidenced  physical  signs  of  tuberculosis,  several  having  symptoms  of  udder 
lesions.     All  of  the  physical  cases  reacted.     (See  Table  6,  herd  8.) 

Herd  No.  9. — A  certified  herd  of  twenty-five  cattle  which  had  been  tested 
semi-annually  by  the  subcutaneous  method  and  found  free  from  reactions  for 
five  years  was  tested  by  the  method  of  Moussu  and  one  cow  reacted.  On  autopsy 
she  was  found  tuberculous.     (For  detailed  description  of  this,  see  cow  1,  p.  129.) 

Herd  No.  10. — Four  out  of  eleven  local  reactors  to  concentrated  tuberculin, 
containing  35  per  cent  glycerin,  injected  intradermally,  failed  later  to  react 
subcutaneously.  Three  of  these  were  autopsied  and  tuberculous  lesions  found 
in  two.  It  is  not  known  whether  the  failure  to  find  lesions  in  the  third  was 
due  to  the  fact  that  only  a  hasty  autopsy  was  made,  or  to  the  fact  that  the 
local  swelling  on  this  cow  was  due  to  the  glycerin  and  not  to  a  reaction.  (See 
cases  2,  5,  and  6,  p.  130.) 

Herd  No.  13. — Thirty-five  cattle  tested  intradermally  with  2  per  cent  pre- 
cipitated tuberculin  gave  fourteen  reactions.  Four  of  these  were  autopsied  and 
all  four  contained  tuberculous  lesions. 

Herd  No.  24. — Thirty-six  cattle  were  tested  after  the  method  of  Moussu. 
One  reacted.  The  entire  thirty-six  were  autopsied  and  no  tuberculous  lesions 
were  found.  It  is  not  known  whether  the  failure  to  find  lesions  in  the  one 
showing  the  local  swelling  was  due  to  the  fact  that  only  a  hasty  autopsy  could 
be  made  or  to  the  fact  that  the  local  swelling  was  caused  by  the  glycerin  and 
was  not  a  reaction. 

Herd  No.  25. — Seventeen  cattle  were  tested  intradermally  with  a  tuberculin 
of  foreign  manufacture.  Four  of  them  reacted.  The  entire  seventeen  were 
autopsied.  Lesions  could  be  found  in  only  three  of  these.  Two  cattle  which 
failed  to  react  were  found  to  have  lesions.  Obviously  the  tuberculin  was 
defective. 


112  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

Herd  No.  29. — Eight  hundred  and  eighteen  cattle  belonging  to  Dairy  Com- 
pany A  were  tested  intradermally,  using  5  per  cent  precipitated  tuberculin, 
and  585  cattle  reacted.  There  has  been  opportunity  to  autopsy  only  seven  of 
these  reactors.  All  but  one  of  them  showed  tuberculous  lesions.  In  addition, 
eight  animals  presumably  physical  cases  of  tuberculosis  were  found  in  the  dairy, 
all  of  which  reacted  to  the  intradermal  test. 

Herd  No.  30-35. — Two  thousand  one  hundred  and  eighty-four  cattle,  belong- 
ing to  Company  B,  were  tested  by  the  intradermal  method,  using  various 
strengths  of  tuberculin,  and  775  reacted.  There  has  been  an  opportunity  to 
autopsy  thirty-two  of  the  reactors,  tuberculous  lesions  being  found  in  all  but 
one  of  these.  Seven  of  the  non-reactors  were  autopsied  and  all  but  one  were 
found  free  from  tuberculous  lesions.  The  lesions  in  the  tuberculous  non-reacting 
animal  were  very  extensive  and  the  failure  of  the  animal  to  react  can  be 
explained  by  the  well-known  fact  that  cattle  having  advanced  tuberculosis 
frequently  are  not  sensitive  to  tuberculin. 


Miscellaneous  Autopsies. — At  various  abattoirs  in  the  San  Fran- 
cisco Bay  region  we  have  applied  the  intradermal  test  to  212  cattle 
and  two  or  three  days  later  have  examined  them  on  the  killing  floor. 
Various  kinds  of  tuberculin  were  used.  The  results  of  these  tests, 
together  with  the  autopsies  of  cattle  from  the  above  mentioned  herds 
are  summarized  in  the  following  table : 


TABLE  2 
Eesults  of  Intradermal  Tests  and  Autopsies 

Cattle  in  which  tuberculous  Cattle  in  which  no  tuber- 
lesions  were  found  culous  lesions  were  found 

Kind  of  Tuberculin  , * s  , *- N 

Number  Number   Per  cent  Number  Number   Per  cent 

tested      reacted      reacted  tested      reacted     reacted 

10  per  cent  Koch's  Old 27  25  92.6  36*  1  2.8 

25  to  100  per  cent  Koch's  Old  2  2  100.0  9  2  22.2 

y2  to  2  per  cent  precipitated....  36  34  94.4  10  1  10.0 

5  to  10  per  cent  precipitated....  46  44  95.6  93  5  5.3 

50  per  cent  precipitated  5  5  100.0  24  2  8.3 

Phymatin 13  13  100.0  23  1  4.3 

Foreign  purified  tuberculin 5  3  60.0  12  1  8.3 

*  These  thirty-six  cattle  were  from  ranges  in  Nevada  where  tuberculosis  is 
rare,  but  there  is  reason  to  believe  that  the  swelling  was  not  a  reaction  but 
due  to  the  glycerin  in  the  tuberculin. 

f  Possibly  not  reactions.  The  swellings  may  have  been  due  to  glycerin  in 
the  tuberculin. 


Bulletin  243] 


INTRADERMAL  TEST  FOR  TUBERCULOSIS 


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Bulletin  243]           INTRADERMAL  TEST  FOR  TUBERCULOSIS  117 

The  autopsy  totals  of  cattle  tested  intradermally  with  10  per  cent 
Koch's  Old  Tuberculin  or  5  to  10  per  cent  alcoholic  precipitated 
tuberculin  are : 

Number  Per  cent 
Number  of  reacting  cattle  in  which  tuberculous  lesions  were 

found  69  94.5 

Number  of  reacting  cattle  in  which  no  tuberculous  lesions  were 

found  4  5.5 

Total  for  reacting  cattle  73  100.0 

Number  of  non-reacting  cattle  in  which  no  tuberculous  lesions 

were  found 123  95.3 

Number   of   non-reacting   cattle   in   which   tuberculous   lesions 

were  found  6  4.7 

Total  for  non-reacting  cattle  129  100.0 


THE  LOCAL  EEACTION  TO  THE  INTRADERMAL  INJECTION  IN  CATTLE 

A  positive  intradermal  reaction  is  indicated  by  a  thickening  of  the 
subcaudal  fold  or  by  the  appearance  at  the  point  of  injection  of  a 
characteristic  sensitive  swelling  varying  in  size  from  that  of  a  small 
pea  to  that  of  an  orange.  The  swelling  may  be  either  soft  and 
oedematous  or  hard  and  inflamed.  A  recognizable  reaction  may  be 
present  on  the  sixth  hour,  but  generally  the  reaction  is  first  clear 
about  the  twelfth  hour  and  continues  to  increase  in  size  until  the 
forty-eighth  hour.  In  some  instances  we  have  observed  that  the  early 
local  reactions  disappear  before  the  forty-eighth  hour,  while  in  others 
delayed  reactions  have  been  noted  which  did  not  appear  until  the 
nineteenth  hour.  In  order  to  be  certain  of  every  case,  observations 
must  be  taken  at  least  twice,  preferably  about  the  thirty-sixth  and 
seventy-second  hours.  If  only  one  observation  can  be  made  it  should 
be  on  the  seventy-second  hour.  Small  indurations  at  the  point  of 
inoculation  about  the  size  of  the  head  of  a  parlor  match  frequently 
occur  in  normal  non-reacting  cattle,  but  anything  larger  than  this 
which  persists  to  the  seventy-second  hour  should  be  considered  a 
positive  reaction. 

In  recording  the  reactions,  a  convenient  method  for  comparison  is 
to  describe  the  size  of  the  swelling  as  pea  size,  hazelnut,  walnut  or 
hen's  egg  size.  The  exact  size,  if  desired,  may  be  measured  by  means 
of  calipers  such  as  are  shown  in  plate  1,  figure  3.    Romer  and  Joseph43 


118  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

consider  that  a  thickening  of  the  skin  fold  more  than  three  millimeters 
larger  than  the  thickness  previous  to  injection  should  be  considered 
a  positive  reaction.  Cases  in  which  the  increase  in  thickness  only 
amounts  to  three  millimeters,  they  consider  doubtful  and  subject  to 
a  retest.  In  our  work,  however,  we  have  found  the  measurement  of 
the  swelling  was  not  of  much  assistance.  Experience  and  practice  will 
enable  the  operator  to  judge  of  the  size  of  the  swellings  without 
measuring,  although  for  the  sake  of  accurate  records  we  have  made 
a  practice  of  taking  careful  measurements. 

In  judging  a  local  swelling,  the  observer  should  depend  more 
upon  the  shape,  appearance,  tenseness,  sensitiveness  and  location  with 
respect  to  the  exact  point  of  injection,  than  upon  the  actual  measure- 
ments. Only  experience  can  teach  an  operator  how  to  be  certain  of 
a  positive  reaction  when  the  local  swelling  is  small.  This  is  especially 
true  when  tuberculin  containing  glycerin  has  been  used. 


THE   THERMAL   REACTION   TO   THE   INTRADERMAL    INJECTION 

IN  CATTLE 

Moussu  and  Mantoux37  state  that  no  thermal  reaction  follows  their 
method  of  performing  the  test.  On  the  contrary,  we  have  found  that 
tuberculous  cattle  usually  do  show  a  marked  temperature  reaction 
when  injected  intradermally  with  the  dosage  and  strength  of  tuber- 
culin recommended  by  these  investigators.  Moussu  and  Mantoux  used 
tuberculin  brut  (Koch's  old  tuberculin)  diluted  with  ten  volumes  of 
physiological  salt  solution.    Their  dose  was  from  %0  to  %  cc. 

We  have  used  this  kind  of  tuberculin  in  testing  207  cattle  when 
frequent  temperature  readings  were  taken  after  the  intradermal 
injection.  One  hundred  and  twenty-four  showed  a  local  reaction  swell- 
ing. In  101  of  these  124  cases  distinct  temperature  reactions  also 
occurred.  Three  cows  which  showed  a  local,  but  no  thermal  reaction, 
were  autopsied  and  found  to  be  tuberculous.  These  cases  are  described 
in  detail  later. 

The  following  table  of  twenty  tests  selected  at  random  from  the 
101  cases  mentioned  above,  shows  the  typical  height  of  the  thermal 
reaction  to  Moussu  and  Mantoux 's  method. 


Bulletin  243] 


INTRADERMAL  TEST  FOR  TUBERCULOSIS 


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UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


In  six  herds  where  we  have  applied  intradermal  tests  to  cattle 
temperatures  were  taken  at  two-hour  intervals  from  the  eighth  to  the 
twentieth  hour  after  the  intradermal  injection,  and,  in  addition,  in 
herd  No.  8  from  the  second  to  the  twentieth  hour  and  in  herd  No.  20 
from  the  eighth  to  the  twenty-eighth  hour. 


TABLE  5 

Showing  Tests  in  Six  Herds  in  which  Thermal  Reactions  Usually 
Resulted  from  an  Intradermal  Injection 


Herd 
No. 

Total 
cows 
tested 

Tuberculin 
used 

Number  of 

cattle  that 

reacted 

locally 

Number  of 

cattle  that 

reacted 

thermally  to 

intradermal 

injection 

Number  of 

cattle  that 

reacted 

locally 

but  not 

thermally 

Number  of 
cattle  that 

reacted 

thermally 

but  not 

locally 

8 

172 

10  per  cent 
0.  T. 

124 

102 

2° 

0 

12 

21 

2  per  cent 
precipitated 

1 

1 

0 

0 

11 

25 

10  per  cent 
O.  T. 

1* 

0 

1 

0 

20 

23 

10  per  cent 
precipitated 

18 

16 

0 

0 

21 

4 

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precipitated 

1 

1 

1 

0 

22 

14 

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precipitated 

6 

6 

0 

0 

22f 

14 

10  per  cent 

4 

4 

1 

0 

precipitated 

Totals 

273 

153 

130 

25 

0 

*  See  autopsy  notes  on. cow  Peggy,  p.  129. 
t  Retest  in  three  days. 


The  following  table  shows  the  results  of  tests  made  on  six  calves 
that  had  been  injected  with  cultures  of  human  tubercle  bacilli  six 
weeks  previous  for  the  purpose  of  immunizing  them  after  the  method 
of  Pearson  and  Gilliland : 


Bulletin  243] 


INTRADERMAL  TEST  FOR  TUBERCULOSIS 


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122  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

It  would  appear  from  this  table  that  the  intradermal  test  has  a 
slightly  depressing  effect  on  the  reaction  to  an  intradermal  injection 
seven  days  later.  The  reaction  swellings  are  uniformly  smaller  and 
the  temperature  does  not  rise  so  high  after  the  second  injection.  The 
animals  described  in  Table  4  were  presumably  healthy  calves  which 
on  June  12,  1913,  had  been  treated  with  cultures  of  human  tubercle 
bacilli,  strain  Revenel  M. 


ACCURACY    OF    THE    INTRADERMAL    COMPARED    WITH    THE 
SUBCUTANEOUS  METHOD 

Investigators  are  so  evenly  divided  on  the  question  of  which  is  the 
more  accurate  test  that  Table  7,  showing  comparative  tests  on  one 
thousand  cattle,  including  326  reactors,  is  worthy  of  study  in  this 
connection.  Moussu  and  Mantoux37  in  their  original  work  com- 
pared the  intradermal  and  subcutaneous  tests  on  seventy  reacting 
animals.  They  found  that  the  two  methods  agreed  in  every  case. 
In  most  instances  they  verified  their  findings  by  autopsy  and  con- 
cluded that  the  intradermal  equals  the  subcutaneous  test  in  accuracy. 
Vallee,  Declaire  and  Herbert48  tested  521  cattle  by  both  methods. 
Four  hundred  and  ninety-four  reacted  to  the  intradermal  and  506  to 
the  subcutaneous;  that  is  twelve  cattle  with  positive  thermal  reactions 
gave  no  intradermal  reaction.  Ward  and  Baker50-51  tested  157  head, 
using  the  10  per  cent  dilution  of  Koch's  old  tuberculin  recommended 
by  Moussu  and  Mantoux.37  They  verified  their  decisions  by  autopsy 
and  concluded  that  the  intradermal  compares  favorably  in  accuracy 
with  the  subcutaneous  method.  Zschocke55  tested  300  cattle  and  found 
that  85  per  cent  of  known  tuberculous  cattle  reacted  to  the  intradermal 
test.  He  considered  the  neck  and  tail  injections  of  equal  value. 
Longley23  and  McKenna26  tested  about  1500  cattle,  using  y2  per  cent 
solutions  of  precipitated  tuberculin,  and  decided  that  the  intradermal 
test  equals  the  subcutaneous  in  accuracy.  Romer  and  Joseph,43  using 
50  per  cent  tuberculin  solutions,  found  that  one  out  of  seventy-nine 
tuberculous  animals  failed  to  react  to  the  intradermal  test.  Norgaard 
and  Case,39  after  testing  several  thousand  head,  decided  that  the  intra- 
dermal test  was  more  satisfactory  than  the  subcutaneous  and  stated 
that  its  substitution  for  the  old  method  was  fully  warranted.  Zwick 
and    Tietze57    admit    no    advantage    for    either    the    intradermal    or 


BULLETIN  243]  INTRADERMAL  TEST  FOR  TUBERCULOSIS  123 

ophthalmic  test  and  have  not  found  them  as  accurate  as  the  sub- 
cutaneous. Tietze47  finds  the  intradermal  more  reliable  than  the 
ophthalmic.  His  conclusion  is  that  the  intradermal  test  is  not  as 
accurate  as  the  subcutaneous.  Lucky,24-25  Sheldon,46  Brown,2  Kinsley 
and  others  in  Missouri  report,  after  having  tested  over  thirty  thousand 
head  by  the  intradermal  method,  that  this  test  is  preferable  under 
average  conditions  when  applied  by  a  skilled  operator.  Hutyra  and 
Marek15  state  that  it  is  desirable  in  practice  where  large  herds  are 
to  be  tested  to  apply  a  local  test  first,  preferably  the  intradermal  or 
ophthalmic  test,  and  after  the  great  majority  of  tuberculous  animals 
have  been  recognized  the  remainder  of  the  herd  may  be  subjected  to 
the  subcutaneous  test.  Martin29  recommends  the  intradermal  test  for 
general  practice.  Melvin,32  of  the  United  States  Bureau  of  Animal 
Industry,  reports  that  the  ophthalmic  and  intradermal  tests  for  the 
detection  of  tuberculosis  in  cattle  have  received  attention.  He  says : 
"These  methods  thus  far  have  not  proved  sufficiently  superior  to  the 
subcutaneous  injection  of  tuberculin  to  warrant  their  general  appli- 
cation in  practice,  although  they  are  still  being  tested  whenever 
suitable  opportunities  are  offered.  A  special  preparation  known  as 
'phymatin'  has  been  used  in  the  ophthalmic  test  and  has  given 
better  results  than  the  alcoholic-precipitated  tuberculin.  The  oph- 
thalmic and  intradermal  methods  of  applying  the  tuberculin  test 
possess  so  many  advantages,  especially  in  the  simplicity  of  reading 
the  results,  that  the  absolute  acceptance  of  either  one  would  be  a  great 
aid  in  the  eradication  of  tuberculosis."  Both  methods  obviate  the 
laborious  operation  of  taking  temperatures  repeatedly  during  the 
day  after  the  injection  and  no  preliminary  temperatures  are  required. 
Littlejohn22  advocates  the  application  of  several  local  tests  simul- 
taneously. Lignieres19  recommends  the  simultaneous  use  of  the  intra- 
dermal, ophthalmic  and  subcutaneous  tests.  Foth11  reports  that  he 
was  able  by  the  aid  of  the  intradermal  method  to  detect  only  about 
one-half  the  tuberculous  individuals  in  Danish  quarantine  cattle.  He 
states  that  the  subcutaneous  injection  has  a  modifying  effect  on  local 
tests  applied  simultaneously.  Assmann1  has  observed  in  certain  cases, 
where  ophthalmic  and  thermal  reactions  occur  at  the  same  time,  that 
the  local  reaction  weakened  as  the  thermal  reaction  gained  strength. 
The  following  table  of  one  thousand  comparative  tests  will  be  of 
interest  to  those  who  desire  information  concerning  the  comparative 
accuracy  of  the  two  methods.  It  will  be  noted  that  of  the  326  cattle 
that  reacted  35  failed  to  react  to  the  intradermal  method  and  44  failed 
to  react  to  the  subcutaneous. 


124 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


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126 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


In  herds  6,  10  and  13  it  is  unfair  to  judge  the  results  from  the 
subcutaneous  test  without  considering  the  possibility  of  a  modifying 
effect  from  the  intradermal  test  just  previous.  We  believe  that  the 
above  results  should  be  interpreted  as  indicating  that  the  two  tests 
are  about  equal  in  accuracy. 

The  results  reported  in  tables  A,  B,  and  C  are  all  by  European 
observers  and  the  proportion  of  reacting  animals  in  which  no  lesions 
were  found  is  much  larger  than  has  been  reported  by  inspectors  in 
the  United  States.  As  we  do  not  know  under  what  conditions  these 
tests  were  performed  no  comparisons  can  be  made  with  the  results 
which  we  have  obtained. 


Klimmer  and  Wolff-Eisner17  in  their  handbook  of  Serumtherapie  and  Serum- 
diagnosis  have  published  the  following  tables  which  are  of  interest  in  a  con- 
sideration of  the  intradermal  method.  It  is  possible  that  some  of  the  figures 
in  these  tables  were  misprints,  as  the  additions  and  percentages  do  not  check 
in  all  cases. 

TABLE   A 


Intradeb 
Veterinarian        Kind  of  tuberculin 
Assman1              Phymatin 

;mal  Tests 

Tuberculous 
animals 

A 

Animals  in  which  no 

lesions  were  found 

on  slaughter 

No. 

tested 
40 

Reacted 
No.    Per  cent 
29           72 

r 

No. 

tested 

11 

Reacted 

No.   Per  cent 

5           45 

Foth11 

Bovotuberkulol   25% 

11 

5 

45 

4 

0 

0 

Bovotuberkulol  50% 

15 

7 

48 

20 

1 

5 

Old  tuberculin    (Hochst)    100% 

15 

8 

52 

45 

13 

29 

Zschocke55 

Phymatin   20% 

46 

39 

84 

53 

11 

21 

Phymatin   50% 

40 

23 

57 

21 

1 

5 

Phymatin    (on  swine) 

14 

13 

93 

82 

1 

1 

Bovotuberkulol  20% 

16 

9 

56 

21 

7 

33 

Bovotuberkulol    (on  swine) 

1 

1 

100 

14 

0 

0 

Tuberkulin   (Marburg) 

20% 

4 

1 

25 

9 

0 

0 

Tuberkulin    (Marburg) 

50% 

18 

9 

50 

23 

7 

30 

Tuberkulin    (on  swine) 

1 

1 

100 

23 

0 

0 

Martin29 

Tuberkulin    (on  swine) 

50% 

10 

10 

100 

93 

0 

0 

Tuberkulin   (on  cattle) 

50% 

33 

33 

100 

17 

1 

6 

Joseph16 

Tuberkulin    (Marburg) 

77 

76 

98 

49 

2 

4.1 

Total 

cattle   

315 

249 

79 

252 

47 

19 

Total 

swine  

26 

25 

96 

212 

2 

] 

TABLE   B 
Results  of  Various  Forms  of  the  Tuberculin  Test  ox  Tuberculous  Cattee 
A  positive  cutaneous      reaction  occurred  in     39  out  of  141  tuberculous  cattle  or  28% 
A  positive  dermal  reaction  occurred  in     38  out  of  143  tuberculous  cattle  or  27% 

A  positive  intradermal  reaction  occurred  in  249  out  of  315  tuberculous  cattle  or  79% 
A  positive  vaginal  reaction  occurred  in     51  out  of     66  tuberculous  cattle  or  77% 

TABLE   C 

Results  of  Various  Forms  of  the  Tuberculin  Test  on  Cattle  in  which  No  Lesions 

Were  Found  on  Slaughter 

A  positive  cutaneous       reaction   occurred  in     6  out  of  125  cattle  or     5% 

A  positive  dermal            reaction   occurred  in      3   out  of  65  cattle   or      5% 

A  positive  intradermal  reaction   occurred   in  47  out  of  252  cattle   or   19% 

A  positive  vaginal            reaction   occurred  in      3    out   of  11  cattle  or   27% 


BULLETIN  243]  INTRADERMAL  TEST  FOR  TUBERCULOSIS  127 

RESULTS  OF  RETESTS 

On  retesting  by  any  form  of  the  tuberculin  test,  a  badly  infected 
herd,  from  which  all  the  reacting  cattle  have  been  removed,  it  will 
usually  be  found  that  from  1  to  30  per  cent  will  react.  This  is  not 
necessarily  due  to  the  failure  of  the  previous  test,  because  the  cattle 
may  in  the  meantime  have  become  infected  f rem  contaminated  pastures 
or  other  sources. 

Another  reason  for  the  reaction  of  such  cattle  on  retest  is  the  fact 
that  cattle  often  do  not  react  during  the  incubation  of  tuberculosis  or 
while  the  disease  is  latent  and  the  lesions  encapsulated.  In  a  badly 
infected  herd  many  animals  may  be  so  recently  infected  that  sensi- 
tiveness to  tuberculin  has  not  developed.  In  tuberculosis  this  period 
of  incubation  is  variable.  In  some  cases  the  disease  probably  begins 
to  progress  on  the  day  of  infection ;  in  others  the  germs  may  be  lodged 
in  the  tissues  for  a  long  time  before  lesions  actually  develop. 

The  arrest  of  the  disease  and  encapsulation  of  the  lesions  is  of 
frequent  occurrence  in  the  tuberculous  cattle.  This  accounts  for  the 
fact  that  cows  which  have  reacted  occasionally  fail  to  react  when 
subsequently  tested.  Later  the  disease  may  start  up  and  then,  on  a 
third  test,  the  animals  may  again  react.  The  commission  appointed  by 
the  American  Veterinary  Medical  Association  have  recognized  these 
facts  in  their  recommendation  that  herds  over  50  per  cent  of  which 
give  positive  reactions  be  handled,  as  far  as  eradication  of  the  disease 
is  concerned,  as  though  all  the  herd  had  reacted. 

The  well-known  fact  that  animals  in  advanced  stages  of  the  disease 
frequently  do  not  react  applies  equally  to  the  intradermal  and  sub- 
cutaneous methods,  but  from  our  observations  we  think  the  former 
will  detect  more  arrested  or  latent  cases.  The  results  in  retesting  com- 
pare favorably  with  those  we  have  obtained  with  the  subcutaneous 
method  under  similar  conditions.  The  following  data  showing  the 
results  of  retests  is  typical  of  what  is  to  be  expected. 


Tests  for  Dairy  Company  B 

Sept.  and  Oct.,  1913.  Tested  by  the  intradermal  method,  2184  cattle,  775  reacted, 
35.5  per  cent.  (Three  hundred  of  the  non-reactors  were  segregated 
on  one  ranch  and  four  months  later  228  of  them  were  retested;  176 
of  the  reacting  cattle  were  also  retested  in  four  months.) 

Feb.,  1913.     Retested  228  non-reacting  cattle,     46  reacted,  20.2  per  cent. 

Feb.,  1913.     Retested  176  reacting  cattle,  173  reacted,  98.3  per  cent. 


No.  con- 

demned 

Per  cent 

14 

18.3 

9 

3.2 

6 

7.2 

32 

22.2 

22 

16.9 

4 

3.1 

•  9 

1.6 

7 

6.1 

4 

4.0 

4 

3.4 

84 

24.r, 

3 

3.4 

..     506 

128  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

Following  are  the  results  of  frequent  subcutaneous  tests  in  three 
dairies  :  DAIEY  No.  1. 

No.  of  cows 
tested 

January  5,  1905  77 

March  12,  1906  62 

September  29,  1906  83 

September  14,  1907  144 

January  29,  1908  130 

August  12,  1908  128 

April  and  September,  1909  125 

January  30,  1910  114 

August  1,  1910 99 

March  21,  1911  118 

Purchases  to  June,  1910  342 

Purchases  since  June,  1910  87 

Total  number  of  cattle  tested  

Total  number  condemned  - 184 

Per  cent  condemned  36.4 

Total  number  of  tests,  including  retests  1,509 

DAIRY  No.  2 

No.  of  cows 
tested 

February,  1908  196 

February,  1909  189 

November,  1909  176 

June,  1910  246 

February  15,  1911  208 

Purchases  to  June,  1910  385 

Purchases  since  June,  1910  14 

Total  number  of  cows  tested  595 

Total  number  condemned  332 

Per  cent  condemned  55.8 

Total  number  of  tests,  including  retests 1,414 

DAIRY  No.  3 

No.  of  cows         No.  con- 
tested              demned  Per  cent 

November,  1908  70                  12  17.1 

April,  1909 57                    9  15.8 

November,  1909  73                    4  5.5 

May,  1910  138                   2  1.5 

November,  1910 198                    6  3.0 

Purchases  to  June,  1910  203                    5  2.5 

Purchases  since  June,  1910  320                    8  2.5 

Total  number  of  cows  tested  593 

Total  number  condemned  48 

Per  cent  condemned 7.8 

Total  number  of  tests,  including  retests  1.059 

In  the  last  three  dairies  the  reacting  cattle  were  at  once  removed 
and  the  barns  were  frequently  disinfected.  Additions  to  the  herd 
were  non-reactors  selected  chiefly  from  infected  herds.  In  the  case 
of  the  cattle  belonging  to  Dairy  Company  B  the  non-reacting  cattle 
were  separated  from  the  reacting,  but  thorough  disinfection  was 
impossible.  Since  the  conditions  in  the  various  dairies  differed 
materially,  no  comparisons  between  the  intradermal  and  subcutaneous 
tests  are  justified. 


No.  con- 
demned 

Per  cent 

110 

56.1 

13 

6.9 

37 

21.0 

18 

7.3 

19 

9.1 

135 

35.1 

Bulletin  243]  intradermal  test  for  tuberculosis  129 

DESCRIPTION    OF    CASES   WHICH   EEACTED    TO    THE    INTRADERMAL 
BUT  DID  NOT  REACT  TO  THE  SUBCUTANEOUS  TEST 

The  following  detailed  history  of  ten  cows  which  reacted  to  the 
intradermal  but  not  to  the  subcutaneous  test  is  submitted  as  proof 
that  cattle,  having  latent,  slight  or  inactive  lesions  of  tuberculosis,  may 
fail  to  react  to  the  subcutaneous  test,  but  may  react  to  the  intradermal. 

Except  when  otherwise  stated,  all  tests  in  these  cases  were  made 
with  Koch's  old  tuberculin,  dilution  1  to  10  of  physiological  salt 
solution.  The  dose  for  the  intradermal  tests  was  y5  cc.  The  dose  for 
the  subcutaneous  tests  varied  from  2  to  4  cc. 

COW  1.  Peggy.  Grade  Holstein.  Good  physical  condition  throughout  the  time 
she  was  under  observation. 

Oct.     10,  1911.     Tested  by  subcutaneous  method.     No  reaction. 

Mar.  2,  1912.  Tested  simultaneously  by  the  subcutaneous  and  intradermal 
methods.  No  thermal  reaction  to  the  subcutaneous.  Strong 
reaction  to  the  intradermal. 

Apr.      2,  1912.     Tested  by  the  intradermal  method.     Strong  reaction. 

July    20,  1912.     Tested  by  the  subcutaneous  method.    No  reaction. 

Oct.       4,  1912.     Tested  by  the  subcutaneous  method.     No  reaction. 

Jan.      1,  1913.     Tested  by  the  intradermal  method.     Strong  reaction. 

Mar.  8,  1913.  Tested  simultaneously  by  the  subcutaneous  and  intradermal 
methods.  No  thermal  reaction  to  the  subcutaneous.  Slight 
reaction  to  the  intradermal. 

July  10,  1913.  Tested  by  the  intradermal  method,  using  y5  cc.  of  10  per  cent 
solution  of  purified  tuberculin.     Slight  reaction. 

July  12,  1913.  This  cow  was  slaughtered  for  beef  under  municipal  inspection. 
One  of  the  retropharyngeal  lymph  nodes  contained  a  tuber- 
culous nodule  five  millimeters  in  diameter  with  a  wall  one 
millimeter  thick  composed  of  greyish  connective  tissue-like 
substance.  In  the  center  was  a  small  amount  of  caseous 
yellow  material,  partially  calcified.  In  one  of  the  mesenteric 
lymph  nodes  a  lesion  very  similar  in  size  and  appearance 
was  found.  No  other  lesions  were  discovered.  A  guinea  pig 
was  inoculated  with  the  tissue  and  on  autopsy  August  13, 
1913,  was  found  to  be  extensively  tuberculous. 

COW  2.     Ear  tag  No.  1649.     Fair  physical  condition. 

May  6,  1913.  Tested  by  intradermal  method.  35  per  cent  O.  T.  tuberculin. 
Positive  reaction. 

May      8,  1913.     Tested  by  subcutaneous  method.    No  reaction. 

June     6,  1913.     Tested  by  ophthalmic  method.     No  reaction. 

June  23,  1913.  Tested  by  ophthalmic  method.  2  per  cent  purified  tuberculin. 
No  reaction. 

June  26,  1913.  Tested  by  the  intradermal  method.  y2  per  cent  purified  tuber- 
culin. Positive  reaction. 


May 

6, 

1913. 

May 

8, 

1913. 

June 

6, 

1913. 

June 

23, 

1913. 

June 

26, 

1913. 

June 

29, 

1913. 

July 

10, 

1913. 

July 

12, 

1913. 

130  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

COW  2 — (Continued). 

June  29,  1913.  Tested  by  the  intradermal  method.  2  per  cent  purified  tuber- 
cu]in.    Positive  reaction. 

July  10,  1913.  Tested  by  the  intradermal  method.  10  per  cent  purified  tuber- 
culin.    Positive  reaction. 

July  12,  1913.  Killed  for  beef  under  federal  inspection.  Four  tuberculous 
nodules  similar  in  appearance  to  those  in  Cow  1  (Peggy) 
were  found  in  the  bronchial  lymph  nodes.  No  other  tuber- 
culous lesions  were  discovered. 

COW  3.     Ear  tag  No.  6.     Aged,  fair  physical  condition. 

Tested  by  intradermal  method.     Positive  reaction. 

Tested  by  subcutaneous  method.    No  reaction. 

Tested  by  the  ophthalmic  method.     Positive  reaction. 

Tested  by  ophthalmic  method.  2  per  cent  purified  tuberculin. 
No  reaction. 

Tested  by  intradermal  method.  V-2  per  cent  purified  tuberculin. 
Positive  reaction. 

Tested  by  intradermal  method.  2  per  cent  purified  tuberculin. 
No  reaction. 

Tested  by  intradermal  method.  10  per  cent  purified  tuberculin. 
Positive  reaction. 

Killed  for  beef  under  municipal  inspection.  The  anterior  and 
posterior  mediastinal  lymph  nodes  were  enlarged  to  three 
times  normal  size  and  contained  dry  calcareous  and  fibrous 
tuberculous  lesions  apparently  inactive  and  of  long  stand- 
ing.    No  other  lesions  were  found. 

COW  4.     Ear  tag  No.  1219. 

May      6,  1913.     Tested  by  intradermal  method.     Positive  reaction. 

May      8,  1913.     Tested  by  subcutaneous  method.     No  reaction. 

June     6,  1913.     Tested  by  ophthalmic  method.     Positive  reaction. 

June  23,  1913.     Tested  by  ophthalmic  method.     2  per  cent  purified  tuberculin. 

No  reaction. 
June  26,  1913.     Tested  by  intradermal  method.     %  per  cent  purified  tuberculin. 

Positive  reaction. 
Tested  by  intradermal  method.     2  per  cent  purified  tuberculin. 

Positive  reaction. 
Tested  by  intradermal  method.     10  per  cent  purified  tuberculin. 

Positive  reaction. 
Killed  for  beef  under  municipal  inspection.     Acute  lesions  of 

tuberculosis  involving  both  anterior  lobes  of  the  lungs  and 

the  mediastinal  and  bronchial  lymph  nodes. 

COW  5.     Ear  tag  No.  1206.     Good  physical  condition. 

May      6,  1913.     Tested  by  intradermal  method.     35  per  cent  O.  T.  tuberculin. 

Strong  positive  reaction. 
May      8,  1913.     Tested  by  subcutaneous  method.    No  reaction. 
June     6,  1913.     Tested  by  ophthalmic  method.    No  reaction. 
June  23,  1913.     Tested  by  ophthalmic  method.     2  per  cent  purified  tuberculin. 

No  reaction. 


June  29, 

1913. 

July  10, 

1913. 

July  12, 

1913. 

Bulletin  243]  intradermal  test  for  tuberculosis  131 

COW  5 — (Continued). 

June  26,  1913.  Tested  by  intradermal  method.  y2  per  cent  purified  tuberculin. 
No  reaction. 

June  29,  1913.  Tested  by  intradermal  method.  2  per  cent  purified  tuberculin. 
Positive  reaction. 

July  10,  1913.  Tested  by  intradermal  method.  10  per  cent  purified  tuberculin. 
Positive  reaction. 

July  12,  1913.  Killed  for  beef  under  municipal  inspection.  No  tuberculous 
lesions  were  found.  An  unusually  careful  search  was  made 
from  a  meat  inspection  standpoint,  but  it  was  impracticable 
to  strip  the  meat  from  the  bones  and  search  the  interior  of 
the  bones  and  joints  and  certain  other  places  where  small 
tuberculous  lesions  might  have  existed. 

COW  6.     Ear  tag  No.  1218. 

May  6,  1913.  Tested  by  intradermal  method.  35  per  cent  O.  T.  tuberculin. 
Positive  reaction. 

May      8,  1913.     Tested  by  subcutaneous  method.     No  reaction. 

June     6,  1913.     Tested  by  ophthalmic  method.  No  reaction. 

June  23,  1913.  Tested  by  ophthalmic  method.  2  per  cent  purified  tuberculin. 
No  reaction. 

June  26,  1913.  Tested  by  intradermal  method.  %  per  cent  purified  tuberculin. 
Positive  reaction. 

June  29,  1913.  Tested  by  intradermal  method.  2  per  cent  purified  tuberculin. 
Positive  reaction. 

July  10,  1913.  Tested  by  intradermal  method.  10  per  cent  purified  tuberculin. 
Positive  reaction. 

July  12,  1913.  Killed  for  beef  under  municipal  inspection.  Soft  caseous  tuber- 
culous lesions  in  the  liver  and  between  the  liver  and  the 
diaphragm.     One  mesenteric  lymph  node  was  caseous. 

COW  7.     Ear  tag  No.  68.     Fair  physical  condition. 

May      6,  1913.     Tested  by  intradermal  method.     2  per  cent  purified  tuberculin. 

Positive  reaction. 
May      8,  1913.     Tested  by  subcutaneous  method.    2  per  cent  purified  tuberculin. 

No  reaction. 
June     6,  1913.     Tested  by  ophthalmic  method.     2  per  cent  purified  tuberculin. 

No  reaction. 
June  23,  1913.     Tested  by  ophthalmic  method.     2  per  cent  purified  tuberculin. 

No  reaction. 
June  26,  1913.     Tested  by  intradermal  method.     y2  Per  cent  purified  tuberculin. 

Positive  reaction. 
June  29,  1913.     Tested  by  intradermal  method.     2  per  cent  purified  tuberculin. 

Positive  reaction. 
July    10,  1913.     Tested  by  intradermal  method.     10  per'cent  purified  tuberculin. 

Positive  reaction. 
July    12,  1913.     Killed  for  beef  under  federal  inspection.     Tuberculous  lesions 

were  present  in  the  anterior  lobes  of  both  lungs  and  in  the 

bronchial  and  retropharyngeal   lymph  nodes. 


132  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

COW  8.     Ear  tag  No.  1213. 

May      6,  1913.     Tested  by  intradermal  method.    Positive  reaction. 

May      8,  1913.     Tested  by  subcutaneous  method.    No  reaction. 

June     6,  1913.     Tested  by  ophthalmic  method.     Positive  reaction. 

June  23,  1913.  Tested  by  ophthalmic  method.  2  per  cent  purified  tuberculin. 
No  reaction. 

June  26,  1913.  Tested  by  intradermal  method.  %  per  cent  purified  tuberculin. 
Positive  reaction. 

June  29,  1913.  Tested  by  intradermal  method.  2  per  cent  purified  tuberculin. 
No  reaction. 

July  10,  1913.  Tested  by  intradermal  method.  10  per  cent  purified  tuberculin. 
Positive  reaction. 

July  12,  1913.  Killed  for  beef  under  municipal  inspection.  Two  small  tuber- 
culous nodules  in  the  liver,  each  about  1  cm.  in  diameter, 
having  firm  fibrous  walls  1  mm.  in  thickness.  Contents  yellow, 
firm,  sprinkled  with  points  of  deeper  yellow  color,  calcification 
was  not  present  and  the  tissue  was  not  entirely  broken  down. 

COW  9.     Ear  tag  No.  1638. 

Jan.    14,  1912.     Tested  by  subcutaneous  method.     No  reaction. 

June     6,  1912.     Tested  by  subcutaneous  method.     No  reaction. 

Jan.      3,  1913.     Tested  by  subcutaneous  method.     No  reaction. 

June     6,  1913.     Tested  by  subcutaneous  method.     No  reaction. 

Tested  by  intradermal  method.     Strong  reaction. 

July  7,  1913.  Autopsied.  In  the  mediastinal  lymph  nodes  were  two  tuber- 
culous lesions  each  about  two-thirds  the  size  of  the  fist, 
encapsulated  and  partially  liquified.  In  the  mesenteric  lymph 
node  was  a  soft  caseous  tuberculous  lesion  two  centimeters 
in  diameter. 

;ag  No.  1220.     Fine  physical  condition  throughout  the  tests. 

Tested  by  intradermal  method.     2  per  cent  purified  tuberculin. 
Positive  reaction. 

Tested  by  subcutaneous  method.     Positive  reaction. 

Tested  by  ophthalmic  method.  Positive  reaction. 

Tested    simultaneously    by   the   subcutaneous    and    intradermal 
methods.    Positive  reaction  to  both  tests. 

Tested  by  ophthalmic  method.     Positive  reaction. 

Tested  by  ophthalmic  method.     Positive  reaction. 

Tested  simultaneously  by  the  ophthalmic,  subcutaneous  and  intra- 
dermal methods.     Positive  reactions  to  the  ophthalmic  and 
intradermal  tests,  no  reaction  to  the  subcutaneous  method. 
Dec.      5,  1913.     Autopsied.     Two  very  small  tuberculous  lesions  in  the  lymph 
nodes  on  rumen. 


Zwick  and  Tietze27  state  that  the  intradermal  and  ophthalmic 
tuberculin  tests  are  of  no  value  in  distinguishing  the  progressive 
cases  from  those  having  arrested  latent  or  healed  lesions.  We  agree 
in  this,  in  that  we  have  not  been  able  to  apply  in  a  practical  way  the 


COW 

10. 

Eai 

May 

6, 

1913, 

May 

8> 

1913 

June 

4, 

1913. 

Aug. 

20, 

1913. 

Oct. 

24, 

1913, 

Oct, 

26, 

1913, 

Dec, 

4, 

1913. 

Bulletin  243]  intradermal  TEST  FOR  TUBERCULOSIS  133 

different  forms  ol  the  tuberculin  test  to  the  classification  of  tuberculous 
cattle  according  to  the  extent  or  activity  of  their  lesions.  However, 
the  cases  which  react  locally  to  the  intradermal  test,  but  which  fail 
to  react  thermally  either  to  the  intradermal  or  subcutaneous  injections, 
have  usually  been  found  on  autopsy  to  contain  small  encapsulated 
lesions. 


DISCUSSION  OF  CASES  WHICH  REACTED   THEEMALLY   TO   THE 

SUBCUTANEOUS  BUT  SHOWED  NO  LOCAL  REACTION 

TO  THE  INTRADERMAL  TEST 

We  have  observed  thirty-five  cases  which  showed  a  thermal  reaction 
to  the  subcutaneous  method  but  no  local  reaction  to  the  intradermal 
method.  This  should  be  considered  in  judging  the  comparative  value 
of  the  intradermal  test.  Fourteen  of  the  above  mentioned  thirty-five 
cows  were  tested  by  both  methods  on  the  same  day.  One  of  these  was 
afterward  autopsied  and  found  to  contain  a  few  active  tuberculous 
lesions.     Following  is  her  history  : 

COW  11,     Ear  tag  No.  2187. 

Jan.      5,  1913.     Tested  by  subcutaneous  method.     No  reaction. 

June  27,  1913.     Tested  by  subcutaneous  method.     Positive  reaction. 
Tested  by  intradermal  method.    No  reaction. 

July  7,  1913.  Autopsied.  The  lungs  contained  one  tuberculous  lesion  the  size 
of  a  half  dollar,  encapsulate  with  caseous  center.  One 
bronchial  gland  contained  a  calcified  tuberculous  lesion  five 
millimeters  in  diameter.  The  liver  contained  two  well-healed 
lesions,  averaging  one  centimeter  in  diameter. 


The  failure  of  this  and  thirty-four  other  cows  to  react  locally 
might  be  accounted  for  by  the  theory  that  a  local  reaction  is  weakened, 
or  disappears,  when  it  occurs  simultaneously  with  a  temperature 
reaction.  The  observations  of  Foth11  and  Assmann1  already  quoted 
are  of  interest  in  this  connection.  However,  we  have  observed  so 
many  instances  in  which  marked  thermal  and  intradermal  reactions 
occurred  simultaneously  from  an  intradermal  injection  that  more 
evidence  is  desirable  before  accepting  this  theory.  "We  are  inclined 
to  attribute  the  failure  of  the  intradermal  test  to  the  well-supported 
theory  that  no  form  of  the  tuberculin  test  will  detect  every  case  of 
tuberculosis  in  cattle,  and  that  the  maximum  number  of  reactors  can 
only  be  determined  by  applying  the  various  tests  simultaneously,  or 
the  local  tests  first  and  following  them  by  the  subcutaneous  method 


134  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

after  an  interval  of  at  least  seven  days.  Assmann1  has  pointed  out 
that  the  proportion  of  questionable,  or  faulty,  diagnoses  amount  to 
over  13  per  cent  for  the  thermal,  while  he  admits  only  9  per  cent  of 
failures  with  the  ophthalmic  test. 

Hastings14  states  that  probably  in  15  per  cent  of  the  animals 
examined  in  badly  infected  herds  the  subcutaneous  test  is  in  error. 
By  far  the  greater  number  of  errors  is  due  to  the  non-reaction  of 
tubercular  animals.  Both  the  thermal  and  intradermal  tests  occasion- 
ally fail  to  produce  reactions  in  tuberculous  cattle  under  circum- 
stances that  exclude  an  explanation  on  the  ground  that  the  disease 
was  either  latent,  or  so  advanced  that  the  cattle  would  not  react. 
The  possibility  of  the  animals  having  been  rendered  insusceptible  by 
previous  injections  of  tuberculin  can  also  be  eliminated. 


THE    COMPAEATIVE    ADVANTAGES   OF   THE    INTRADERMAL    AND 
OPHTHALMIC  METHODS  IN  TESTING  CATTLE 

Six  state  veterinarians  have  recently  written  us  that  they  consider 
the  intradermal  method  preferable  to  the  ophthalmic.  Hutyra  and 
Marek15  also  indicate  a  preference  for  the  intradermal  over  the 
ophthalmic.  Our  data  on  this  point  are  rather  meager.  In  tests  on 
known  tuberculous  animals  with  10  per  cent  solutions  of  Koch's  old 
tuberculin  our  results  were  unsatisfactory,  being  negative  in  all 
instances.  Five  per  cent  solutions  of  precipitated  tuberculin,  however, 
proved  very  satisfactory.  In  139  cattle  known  to  be  reactors  to  the 
subcutaneous  or  intradermal  methods,  when  tested  by  the  ophthalmic 
method,  using  this  tuberculin,  we  produced  pronounced  conjunctival 
reactions  in  every  case.  In  one  instance  a  cow  reacted  to  the 
ophthalmic   test,   but   failed   to   react   to   either   the   intradermal    or 


*  The  ophthalmic  tuberculin  test  as  first  applied  to  cattle  in  1907  did  not  give 
very  uniform  results,  probably  because  it  was  made  either  with  a  weak  solution 
or  with  tuberculin  diluted  with  irritating  solutions  of  glycerin,  trikresol,  or 
phenol.  Recent  observations  by  Foth11  and  others  have  shown  that  5  per  cent 
solutions  of  dry  tuberculin  give  very  good  results  in  this  test. 

Technic  of  the  Ophthalmic  Test. — An  assistant  should  grasp  the  animal  by  the 
nose  and  turn  the  head  at  an  angle  so  that  the  operator  may  drop  one  or  two 
drops  of  the  tuberculin  directly  upon  the  eyeball.  A  small  hypodermic  syringe 
is  more  suitable  for  this  purpose  than  a  medicine  dropper.  The  closed  eye  is  then 
slightly  massaged  for  several  seconds.  In  cattle  the  reaction  usually  sets  in 
by  the  eighth  hour.  It  manifests  itself  by  a  watering  of  the  eye,  reddening 
of  the  conjunctiva  and  dropsical  swelling  of  the  lid.  Later  a  purulent  exudate 
accumulates  at  the  inner  corner  of  the  eye  and  usually  runs  down  the  face. 
This  sometimes  dries  up  and  drops  off  in  a  few  hours,  so  that  the  evidences  of 
the  reaction  may  entirely  disappear  in  twenty-four  hours,  although  occasionally 
they  can  be  seen  for  several  days.  Although  the  results  of  the  test  are  usually 
apparent  by  the  twelfth  hour,  additional  observations  should  be  made  on  the 
eighteenth  and  twenty-fourth  hours  to  detect  any  delayed  reactions. 


BULLETIN  243]  INTRADERMAL  TEST  FOR  TUBERCULOSIS  135 

subcutaneous  tests.  On  autopsy  this  cow  proved  tuberculous.  In 
testing  range  cattle  we  have  found  it  easier  to  apply  the  intradermal 
than  the  ophthalmic  test.  The  eye  test  is  more  reliable  than  either 
the  intradermal  or  subcutaneous  methods  for  testing  animals  which 
have  been  recently  injected  with  tuberculin.  The  test  can  be 
repeatedly  applied  without  injury  to  the  eye.  Its  frequent  repetition 
does  not  injure  its  accuracy.  When  given  simultaneously  with, 
or  subsequent  to,  either  the  intradermal  or  subcutaneous  test  it 
may  occasionally  assist  in  deciding  doubtful  reactions.  Since  the 
reaction  usually  occurs  by  the  eighth  hour,  it  is  sometimes  found 
preferable  in  veterinary  practice  to  use  this  method  when  quick 
results  are  necessary.  Our  attempts  to  use  the  ophthalmic  method 
in  the  routine  testing  of  dairy  cows  have  failed  to  give  satisfaction. 
In  one  instance  when  the  test  was  applied  to  a  dairy  of  eighty-six 
cows,  known  to  include  at  least  forty  reacting  animals,  the  rain 
washed  out  or  obscured  all  but  one  conjunctival  reaction.  In  other 
instances  we  have  observed  that  the  exposure  to  bright  sunshine  and 
wind  has  caused  equally  unsatisfactory  results.  The  test  is  only 
satisfactory  when  the  cattle  can  be  kept  tied  or  confined  in  stanchions 
under  shelter  for  a  period  of  at  least  sixteen  hours,  except  in  the  case 
of  animals  which  show  a  reaction  before  that  time.  The  cattle  should 
be  fed  only  on  concentrates,  or  finely  chopped  hay.  We  have  verified 
the  observation  of  White  and  McCampbell  that  it  is  advisable  not  to 
feed  long  hay  while  the  reaction  is  developing  on  account  of  the 
possibility  of  wiping  off  the  exudate.  Klimmer,17  Assman,1  Melvin,32 
Konge18  and  others  have  reported  excellent  results  with  the  ophthalmic 
test  when  using  a  preparation  known  as  phymatin.  In  testing 
twenty-three  tuberculous  cattle  with  this  preparation  we  have  obtained 
positive  reactions  in  every  case.  Phymatin  will  also  produce  reactions 
when  injected  intradermally. 

USE  OF  THE  INTRADERMAL  TUBERCULIN  TEST  ON  SWINE 
At  the  University  of  California  Hog  Serum  Laboratory  the  intra- 
dermal test  is  used  as  a  matter  of  routine  on  all  hogs  purchased.  The 
test  is  very  satisfactory,  the  reactions  usually  being  larger  in  size 
than  in  cattle.  A  dose  of  at  least  %  cc-  should  be  used.  We  are 
undecided  as  to  the  best  place  to  make  the  injection.  Injections  into 
the  skin  at  the  edge  of  the  ear  are  very  easily  seen.  However,  when 
the  injection  is  made  at  the  base  of  the  ear  the  swellings  are  often 
larger.  Tuberculous  hogs  often  show  a  thermal  as  well  as  a  local 
reaction  to  %  cc.  doses  of  a  10  per  cent  solution  of  alcoholic  pre- 
cipitated tuberculin. 


136  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

Following  is  a  statement  of  certain  tests  made  by  Dr.  J.  F.  Mitchell 
at  the  University  of  California  Hog  Serum  Laboratory:  Sixty-nine 
hogs  were  tested  by  the  intradermal  method,  using  10  per  cent  Koch's 
Old  Tuberculin,  and  fifteen  reacted.  Three  days  later  the  ophthalmic 
test  was  administered,  using  the  same  kind  of  tuberculin.  No  eye 
reactions  occurred,  except  possibly  in  one  case,  which  showed  a  very 
slight  discharge  from  one  eye.  Twenty  days  later  sixty-two  of  these 
hogs,  including  the  fifteen  reactors,  were  again  tested  by  the  intra- 
dermal method  and  twenty  reacted.  We  used  2  per  cent  precipitated 
tuberculin  on  nineteen  of  these,  including  four  that  had  previously 
reacted.  Six  reactions  resulted  with  this  tuberculin  including  the 
four  hogs  which  had  reacted  at  the  previous  test.  Ten  per  cent  Koch's 
Old  Tuberculin  was  used  on  the  remaining  forty-three,  including  nine 
hogs  that  had  reacted  at  the  previous  test,  and  fourteen  of  the  forty- 
three  reacted,  including  the  nine  that  had  previously  reacted. 

Fourteen  days  later  eighteen  of  the  reacting  hogs  were  given  the 
ophthalmic  test,  using  5  per  cent  precipitated  tuberculin,  and  seven 
of  these  showed  a  very  slight  yellowish  discharge  in  the  eye  injected, 
but  not  enough  to  be  of  any  great  diagnostic  value.  Nine  of  the 
reactors  have  been  killed,  and  in  one  of  them  we  failed  to  find  lesions ; 
the  others  were  tuberculous.  In  only  one  case  was  the  entire  carcass 
condemned  for  tuberculosis. 


CONDITIONS  UNDER  WHICH  THE  INTRADERMAL   TEST 
IS    PREFERABLE 

Since  the  reaction  to  tuberculin  when  injected  by  the  intradermal 
method  depends  on  a  swelling  at  the  point  of  injection  and  not  on 
thermal  manifestations,  it  is  to  be  preferred  to  the  subcutaneous 
injection  under  all  conditions  that  are  liable  to  modify  the  tuberculin 
temperature  curve.  In  testing  wild  range  cattle,  even  if  it  is  possible 
to  restrain  them,  no  reliance  can  be  placed  on  the  reaction  as  shown 
by  the  tuberculin  temperature  curve. 

In  calves  under  six  months  of  age  there  is  liable  to  be  a  difference 
of  two  or  more  degrees  in  the  normal  temperature  on  two  consecutive 
days.  The  temperature  of  some  cows  is  easily  influenced  by  variations 
in  the  weather  and  also  by  changes  in  handling  and  feeding.  We  deem 
it  advisable  to  test  by  the  intradermal  method  if  the  temperature  of 
the  air  where  the  cattle  are  confined  exceeds  100  degrees  F.  or  in  cold 
weather  if  the  animals  will  be  compelled  to  stand  in  a  draught. 
Cattle  from  a  long  journey  by  road  or  rail  are  apt  to  have  an  abnormal 
temperature. 


Bulletin  243]  INTRADERMAL  TEST  FOR  TUBERCULOSIS  137 

If  an  animal  reacts  locally  there  is  a  record  left  at  the  point  of 
injection  in  the  form  of  a  swelling,  this  swelling  being  discernible  in 
some  instances  three  weeks  after  injection. 

The  local  intradermal  reaction  is  not  affected  by  advanced 
pregnancy,  recent  parturition  or  the  period  estrum.  We  hesitate  to 
mention  this  as  an  advantage  over  the  subcutaneous  method,  however, 
for  in  our  experience  in  testing  numerous  cows  in  this  condition  we 
have  not  observed  that  the  reliability  of  the  subcutaneous  test  was 
affected. 

Although  we  have  reason  to  believe  that  the  intradermal  method 
modifies  to  some  extent  the  results  of  a  subsequently  performed  sub- 
cutaneous test,  its  effect  is  certainly  not  so  pronounced  as  a  previous 
subcutaneous  test.  When  cattle  are  to  be  retested  a  weak  solution 
(i/2  per  cent  precipitated  tuberculin)  can  be  used.  We  believe  that 
the  intradermal  test  can  always  be  satisfactorily  substituted  for  the 
subcutaneous  by  an  experienced  operator,  provided  a  strong  tuber- 
culin (5  per  cent  or  stronger  solution  of  precipitated)  is  used  for  the 
intradermal  injection  and  daily  observations  on  the  swellings  are 
continued  to  the  ninetieth  hour.  In  fact,  we  are  convinced  that  when 
performed  in  this  way  on  large,  badly  infected  herds  more  tubercular 
animals  will  be  detected  than  when  dependence  is  placed  on  tempera- 
ture reaction  alone. 

It  is  reported  that  in  certain  pure  bred  herds  where  the  owners 
permit  prospective  purchasers  to  apply  the  tuberculin  test  the  animals 
sometimes  receive  several  tests  a  year.  The  substitution  of  either  the 
intradermal  or  ophthalmic  test,  preferably  the  latter,  would  be 
desirable  in  such  herds,  because  these  tests,  not  being  so  liable  to 
render  the  infected  animals  insensitive  to  tuberculin,  would  have  the 
advantage. 


CONDITIONS   UNDER    WHICH    THE    INTRADERMAL    TEST    IS 

UNRELIABLE 

Tuberculous  cattle  that  have  been  recently  injected  with  tuberculin 
frequently  do  not  respond  to  the  intradermal  test,  The  method  does 
not  seem  to  have  much  advantage  over  the  subcutaneous  in  this  respect, 
and  is  inferior  to  the  ophthalmic  for  retesting.  Repeated  intradermal 
tests  render  tuberculous  cattle  unresponsive  to  either  the  subcutaneous 
or  intradermal  methods. 

In  the  hands  of  an  inexperienced  man  the  intradermal  is  not  so 
reliable  as  the  subcutaneous  or  ophthalmic  methods.    Even  a  graduate 


138  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

veterinarian  who  has  not  received  special  instruction  or  at  least  per- 
fected himself  in  the  interpretation  of  the  local  swellings  by  several 
autopsies  or  observations  of  this  test  when  administered  in  conjunc- 
tion with  the  usual  subcutaneous  method,  in  our  opinion  is  not  capable 
of  securing  good  results. 

Since  small  doses  are  used  in  the  intradermal  method  it  is  of  the 
highest  importance  that  the  tuberculin  bu  of  a  high  standard  of 
potency.  It  must  be  sterile  and  free  from  glycerin.  It  must  be 
administered  in  a  clean  and  skillful  manner.  A  bungling  operator, 
by  introducing  the  needle  several  times  may  cause  traumatic  injuries 
which  may  have  the  appearance  of  reactions.  Observations  on  the 
reactions  should  be  continued  until  the  seventy-second  hour.  Under 
no  circumstances  can  this  test  be  finished  as  quickly  as  the  other 
methods.  If  pressed  for  time  the  veterinarian  must  depend  on  the 
ophthalmic  and  subcutaneous  method.  Dr.  Law  in  his  treatise  on 
veterinary  medicine,  mentioned  seventeen  conditions  under  which  he 
believes  the  subcutaneous  method  to  be  unreliable,  but  in  most  instances 
these  do  not  apply  to  the  intradermal  method.  In  competent  hands 
this  method  has  the  advantage  over  either  the  subcutaneous  or  ophthal- 
mic under  average  California  conditions. 

Cattle  in  a  very  thin,  starved  condition  are  not  suitable  subjects 
for  any  form  of  the  tuberculin  test.  In  testing  some  dry  dairy  cows 
that  had  been  turned  out  on  scanty  pasture,  we  observed  that  several 
known  tuberculous  animals  failed  to  react  to  the  subcutaneous  test. 
The  preliminary  results  of  experiments  which  we  are  making  on  some 
stock  indicated  that  during  the  winter,  when  the  feed  is  poor  and  the 
cattle  are  somewhat  reduced  in  flesh,  their  sensitiveness  to  tuberculin 
is  not  as  pronounced  as  when  they  .are  in  prime  condition. 

ANSWERS  TO  CERTAIN  OBJECTIONS  TO  THE  INTRADERMAL  METHOD 

An  argument  that  has  been  advanced  against  the  use  of  the  intra- 
dermal method  for  official  tuberculin  testing  is  that  tabulated  records 
of  the  test  results  cannot  be  made.  Would  not  a  tabulation  of  the 
measurements  of  the  swellings,  together  with  notes  on  their  appear- 
ance and  sensitiveness,  constitute  an  acceptable  test  record?  Vallee49 
suggests  that  a  series  of  observations  be  expressed  graphically.  An 
advantage  of  the  intradermal  test  in  this  respect  is  that  reacting 
swellings  usually  persist  for  several  days  and  in  some  cases  for  weeks. 
The  presence  or  absence  of  such  a  swelling  in  case  of  dispute  would 
constitute  evidence  which  would  add  to  the  value  of  the  tuberculin 
test  record.      We  have  observed  that  some  cattle  which  reacted  to  the 


BULLETIN  243]  INTRADERMAL  TEST  FOR  TUBERCULOSIS  139 

intradermal  showed  a  return  of  the  local  reaction  when  retested  by 
the  subcutaneous  method.  Occassionally  this  recurring  local  reaction 
has  exceeded  the  original  in  size.  This  return  of  the  subcaudal  swell- 
ing after  the  injection  of  tuberculin  subcutaneously  in  the  neck  has 
been  observed  even  after  an  interval  between  tests  of  three  months. 

Another  objection  is  that  it  requires  an  expert  and  experienced 
man  to  use  the  intradermal  method  successfully.  We  admit  that 
the  test  demands  some  experience,  but  have  pointed  out  that  any 
practitioner  can  gain  that  experience  while  applying  the  usual  sub- 
cutaneous method.  On  the  other  hand,  it  is  not  a  test  which  can  be  as 
readily  used  by  the  layman  as  the  subcutaneous  method.  We  mention 
this  latter  fact,  knowing  that  it  may  appeal  to  many  as  a  favorable 
argument,  although  it  is  to  be  regretted  that  there  has  not  as  yet  been 
perfected  a  test  so  simple  and  accurate  that  any  dairyman  can  use  it 
on  his  own  herd.  We  believe  that  the  ophthalmic  comes  nearer  to  this 
than  either  the  intradermal  or  subcutaneous  method. 

The  very  just  objection  is  made  that  as  yet  we  do  not  know  whether 
the  test  is  as  accurate  as  the  subcutaneous  method.  More  data  are 
needed  on  this  point.  Comparison  should  be  made  by  autopsy,  and 
I  regret  that  in  California  we  have  been  able  to  follow  up  with 
autopsies  only  341  cases  out  of  4001  tested.  To  be  of  any  value  such 
autopsies  should  be  made  on  the  non-reacting  as  well  as  reacting 
animals.  Most  tests  that  have  been  recently  made  to  determine  the 
value  of  the  intradermal  have  included  the  autopsy  of  both  the 
reacting  and  non-reacting  animals.  The  intradermal  test  is  so  easy 
to  apply  that  it  would  be  possible  for  federal  or  state  officials  to 
arrange  with  abattoirs  to  apply  the  test  to  thousands  of  head  and 
follow  them  to  the  killing  floor.  Titze47  has  concluded  after  a  study 
of  the  results  of  the  intradermal  and  ophthalmic  tests  in  Germany 
that  these  methods  are  not  equal  in  accuracy  to  the  subcutaneous. 
Quoting  statistics  of  the  U.  S.  Bureau  of  Animal  Industry,31  he  assumes 
that  it  is  possible  to  make  a  sure  diagnosis  by  the  subcutaneous  method 
in  more  than  97  per  cent  of  tuberculous  cattle.  Hastings14  has  pointed 
out  the  fallacy  of  such  deductions.  Although  98.93  per  cent  of  the 
since  no  autopsies  were  made  on  them.  We  believe  that  the  sub- 
under  supervision  had  tuberculous  lesions,  it  is  not  known  what 
proportion  of  the  363,008  non-reacting  cattle  really  had  tuberculosis. 
24.784  reacting  cattle  reported  by  Melvin31  as  having  been  autopsied 
cutaneous  test  as  ordinarily  performed  on  large  numbers  of  cattle 
will  fail  to  detect  tuberculosis  in  at  least  10  per  cent  of  the  occult 
cases.  Wills54  found  with  the  subcutaneous  test  that  over  20  per  cent 
of  the  thermal  reactions  in  seventy-two  tuberculous  cattle  occurred 


140  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

subsequent  to  the  eighteenth  hour  after  injection.  Even  if  it  is  event- 
ually found  that  the  intradermal  test  fails  to  detect  tuberculosis  in 
10  or  15  per  cent  of  occult  cases,  we  would  still  consider  it  as  efficient 
as  the  subcutaneous  method. 

The  strongest  objection  to  the  intradermal  test  seems  to  be  that 
as  yet  a  standard  tuberculin  for  this  test  has  not  been  perfected.  With 
the  six  leading  biological  houses  of  this  country  each  putting  out  a 
different  kind  of  "intradermal  tuberculin,"  some  of  which,  to  say 
the  least,  are  unsuitable  for  the  purpose,  uniform  results  are  impossible. 

We  have  advocated  in  this  paper  the  combination  of  the  intra- 
dermal and  ophthalmic  with  the  subcutaneous  method.  The  objection 
is  that  this  requires  too  much  time  and  work.  This  is  the  crux  of 
the  whole  bovine  tuberculosis  situation.  Such  objections  are  made 
by  practitioners  who  are  willing  to  slight  their  work  in  order  to 
obtain  an  easy  fee.  In  case  it  is  impossible  to  perform  the  sub- 
cutaneous test  thoroughly,  the  intradermal  should  certainly  be  used. 
Sehniirer45  has  pointed  out  that  the  combination  of  several  tests 
increases  the  certainty  of  the  results.  The  combined  tests  add  accuracy 
by  confirming  the  many  questionable  reactions  that  occur  and  by 
detecting  tuberculous  cows  that  react  to  but  one  test.  At  present  it 
would  be  a  backward  step  to  abandon  the  subcutaneous  method  for 
official  work.  Combining  the  two  tests  gives  time  for  physical  exami- 
nation, in  Avhich  more  thoroughness  is  sadly  needed  in  this  state. 


SUMMARY 

In  all.  4926  intradermal  tests  have  been  made  on  4001  cattle;  1614 
of  which  reacted,  and  there  has  been  an  opportunity  to  check  these 
results  by  autopsies  in  341  cases  and  by  the  discovery  of  lesions  on 
physical  examination  in  54  additional  cases. 

To  291  cattle  that  reacted  to  the  intradermal  test  the  subcutaneous 
test  was  subsequently  or  simultaneously  applied,  and  282  cattle  reacted. 
Forty-four  cattle  with  a  positive  intradermal  reaction  gave  no  thermal 
reaction  to  the  subcutaneous  test.  It  was  possible  to  autopsy  only 
ten  of  these  which  failed  to  react  to  the  subcutaneous  method,  but 
in  nine  of  the  ten  tuberculous  lesions  were  found.  Thirty-five  cattle 
which  failed  to  react  to  the  intradermal  test  subsequently  reacted  to 
the  subcutaneous  test.  Two  of  these  cows  which  reacted  to  the  sub- 
cutaneous method,  but  showed  no  swelling  at  the  point  of  intradermal 
injection,  were  found  on  autopsy  to  be  tuberculous. 


Bulletin  243]  intradermal  test  for  tuberculosis  141 

Cattle  which  react  to  the  intradermal  test  with  local  swelling 
usually  have  also  a  thermal  reaction  similar  to  that  produced  by  the 
ordinary  subcutaneous  injection. 

The  intradermal  test  is  unreliable  when  applied  a  few  days  after 
a  subcutaneous  injection.  The  time  limits  under  which  this  holds  true 
have  not  been  determined. 

The  intradermal  test  appears  to  have  some  modifying  effect  on  a 
subsequent  test  by  either  the  intradermal  or  subcutaneous  methods,  but 
this  is  not  so  pronounced  as  that  produced  by  the  usual  subcutaneous 
injection. 

The  thermal  reaction  which  accompanies  the  intradermal  injection 
usually  subsides  in  twenty  hours,  but  it  seems  desirable  not  to  apply 
the  subcutaneous  test  for  several  days  after  the  intradermal.  The 
longer  the  interval  the  better. 

The  subcaudal  fold  is  a  more  suitable  point  for  the  intradermal 
injection  than  the  neck. 

Our  results  with  Koch's  Old  Tuberculin  in  10  per  cent  solutions 
and  with  alcoholic  precipitated  tuberculin  in  y2  per  cent  solutions 
compare  favorably  in  accuracy  with  the  usual  subcutaneous  method. 
A  more  satisfactory  method,  however,  is  to  use  %  cc.  intradermal 
doses  of  alcoholic  precipitated  tuberculin  in  5  to  50  per  cent  solutions 
administered  subcaudally,  and  2  cc.  doses  of  10  per  cent  Koch's  Old 
Tuberculin  administered  subcutaneously,  and  take  temperatures  as  in 
the  usual  subcutaneous  test.  With  this  kind  and  strength  of  tuberculin 
the  local  reactions  are  pronounced  and  the  thermal  reactions  are  as 
characteristic  as  those  that  occur  from  subcutaneous  injections  alone. 

The  procedure  in  injecting  cattle  is  not  difficult,  provided  needles 
of  the  proper  length  are  used.  To  obtain  the  best  results  the  tuber- 
culin must  be  injected  into  the  lower  layers  of  the  skin  or  into  the 
subdermal  connective  tissue.  The  application  of  strong  disinfectants 
to  the  site  of  injection  should  be  avoided.  It  requires  considerable 
experience  to  correctly  interpret  the  local  swellings.  This  is  especially 
true  when  tuberculin  containing  glycerin  is  used. 

We  do  not  advocate  the  substitution  of  the  intradermal  for  the 
subcutaneous  method  by  a  veterinarian  until  he  has  become  skilled  in 
its  use  by  practice  and  observation.  The  practitioner  can  easily 
accomplish  this  in  the  routine  of  his  work  by  applying  the  two  tests 
simultaneously.  Such  a  procedure  cannot  injure  the  accepted  sub- 
cutaneous method  in  any  way,  although  it  may  modify  the  local 
intradermal  swellings  to  some  extent. 

Our  observation  of  the  fact  that  cattle  having  small,  latent,  or 
arrested  lesions  will  sometimes  react  to  the  intradermal  and  not  to  the 


142  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

subcutaneous  test  suggests  the  possibility  of  differentiating  between 
latent  and  acute  infections  by  combining  the  two  tests,  but  we  have 
not  been  able  to  put  this  into  practical  use. 

When  5  per  cent  solutions  of  potent  precipitated  tuberculin  are 
used  both  the  ophthalmic  and  intradermal  methods  equal  the  sub- 
cutaneous test  in  accuracy,  but  we  have  found  the  ophthalmic  method 
unsuitable  for  use  in  routine  testing  under  California  conditions, 
except  when  the  cattle  can  be  kept  indoors. 

The  intradermal  method  has  proven  very  satisfactory  in  the  out- 
of-door  tuberculin  testing  frequently  necessary  in  this  state  and,  for 
the  non-official  testing  of  large  numbers  of  cattle,  is  preferable  to  the 
subcutaneous  method.  It  requires  more  experience  to  apply  the  test 
and  interpret  the  intradermal  reactions  than  is  required  in  testing  by 
the  subcutaneous  or  ophthalmic  methods. 

Some  tuberculous  cattle  react  locally  to  the  intradermal  test,  but 
fail  to  react  thermally  to  the  subcutaneous  test,  while  others  which 
react  thermally  fail  to  react  locally.  Obviously,  then,  it  is  necessary 
to  use  both  tests  in  order  to  detect  the  maximum  number  of  reacting 
cattle.  By  administering  the  ophthalmic,  intradermal  and  sub- 
cutaneous tests  simultaneously,  a  higher  percentage  of  tuberculous 
cattle  will  be  detected  than  if  dependence  is  placed  on  one  test  alone. 

The  intradermal  test  is  especially  adapted  to  the  testing  of  swine 
and  has  proven  of  value  in  the  routine  work  of  the  California  Hog 
Serum  Laboratory. 


Bulletin  243]  intradermal  test  for  tuberculosis  143 


REFERENCES 

i  Assman,  W.:  Comparative  investigations  on  the  thermal  tuberculin  test  and 
phymatic-ophthalmo  reaction.     Berlin.     Tierarztlich.  Wochensch.,  vol.  27 
(1911),  no.  25,  p.  449. 
2  Brown,  L.  D.:   Editorial,  Amer.  Jour.  Yet.  Med.,  vol.   7,  no.  7    (July,  1912), 

pp.  305-306. 
s  BilCHLi,  K.:  Die  Klinik  und  die  Bekampfung  der  Rindertuberkulose.     Inau. 

Dis.,  Bern,  1909. 
4  Case,  L.  N.:  Tuberculosis  Control.     The  Hawaiian  Forester  and  Agriculturist, 

vol.  10,  no.  5,  p.  129. 
s  Christiansen,    M.:    The    intracutaneous    test    for    detecting    tuberculosis    in 
bovines.     Abs.  in  Berlin.     Tierarztlich.  Wochensch,  vol.  27  (1911),  no.  28, 
pp.  509-510.     Expt.  Sta.  Record,  vol.  26,  no.  6  (May,  1912),  p.  583. 
e  Corbett,  L.,  and  Griffith,  A.  S. :  Report  of  Royal  Com.  on  Tuberculosis.    Part 
2,  appendix.     Abs.  in  Jour.  Comp.  Path,  and  Ther.,  vol.  26,  part  2,  p.  199 
(June,  1912). 
7  de  Bliek:  Citation  by  Ostertag,  see  ref.  40. 

s  Brieger  and  Proskauer:  Citation  by  Hutyra  and  Marek,  see  ref.  15. 
9  Editorial,  Amer.  Vet.  Review,  vol.  34,  p.  570.     Intradermo  reaction. 

io  Editorial,  Amer.  Vet.  Review,  vol.  34,  pp.  570-571.  Value  of  Tuberculin  Re- 
actions. 

hFoth:  Tuberculinproben  nach  Moussu  and  Mantoux.  Berlin.  Tierarztlich. 
Wochensch.,  1909,  p.  727. 

12  Hamman,  L.,  and  Wolman,  S.:  Tuberculin  in  Diagnosis  and  Treatment.  (D. 
Appleton  &  Co.,  1912),  pp.  151-153. 

is  Haring,  C.  M.:  Bovine  Tuberculosis  Investigations  at  the  University  of  Cali- 
fornia Farm.  Proceedings  of  the  American  Veterinary  Medical  Associa- 
tion, 1910,  pp.  306-314. 

14  Hastings,  E.  G.:  The  Limitations  of  the  Tuberculin  Test.  American  Veterin- 
ary Review,  vol.  42,  no.  4  (Jan.,  1913),  pp.  384-398. 

is  Hutyra  and  Marek:  Pathology  and  Therapeutics  of  Diseases  of  Animals,  vol. 
1,  pp.  584-586. 

is  Joseph,  K. :  The  Intracutaneous  Tuberculin  Test.  Berlin.  Tierarztlich.  Woch- 
ensch., vol.  25,  no.  46,  pp.  847-851. 

it  Klimmer,  M.,  und  Wolff-Eisner:  Tuberkulosediagnostik  mit  Tuberkulin- 
praparaten.  Handbuch  der  Serumtherapie  und  Serumdiagnostik  in  Vet- 
erinarmedizin,  1911,  pp.  111-122. 

is  Konge,  W.:  Uber  den  Wert  der  Tuberculinaugenprobe.  Berlin.  Tierarztlich. 
Wochensch.,  vol.  29,  no.  45,  pp.  800-801. 

19  Lignieres,  J.:  Nouvelle  Contribution  a  1 'etude  des  reactions  locales  a  la  tuber- 
culin.    Bull.  Soc.  Cent,  de  Med.  Veterinaire,  vol.  63,  pp.  146-167. 

20 Tuberculosis   and   Tuberculin.      Bull.    Soc.   Cent.    Med.   Vet.,    vol.    88 

(1911),  no.   20,  pp.  433-436.     Abs.   in   Expt.   Sta.   Record,  vol.   26,   no.   8 
(June,  1912). 

2i Associated    Tuberculin    Tests.      Journ.    of    Comp.    Path    and    Ther. 

(March,  1909),  vol.  22,  no.  1,  p.  233. 

22  Littlejohn,  A.  R.:    Intradermal  Reaction.     Vet.   Journ.,  vol.   67    (1911),  pp. 

343-344. 

23  Longley,  Otis  A.:  The  Intradermal  Tuberculin  Tests.     Mulford's  Veterinary 

Bulletin,  vol.  4,  no.  2  (July,  1912),  pp.  52-55. 
24Luckey,  D.  F.:   Amer.  Journ.   Vet,  Med.,   March,   1912,  p.   81;    Discussion   in 
April,  1912,  number,  p.  144. 


144  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 

25  Luckey,  D.  F. :    The  Intradermal  Test.    Amer.  Vet.  Rev.,  vol.  41,  no.  3   (June, 

1912),  pp.  316-323. 

26  McKenna,  J.  F.:    The  Intradermal  Tuberculin  Test.     Mulford's  Veterinary 

Bulletin,  vol.  5,  no.  1  (April,  1913),  pp.  5-8. 

27  Mantoux  and  Eoux:  Citation  by  Hamman  and  Wolman,  see  ref.  12. 

28  Mantoux  and  Lemaire  :  Citation  by  Hamman  and  Wolman,  see  ref.  12. 

29  Martin,  G.:  Practical  Results  with  the  Intracutaneous  Tuberculin  Reaction 

with  Hogs  and  Bovines.  Beitr.  Klinik  Tuberkulose,  vol.  16  (1909),  no.  1, 
pp.  37-54.    Abs.  in  Expt.  Sta.  Record,  vol.  26,  no.  2  (February,  1912). 

30  Matschke,  J.:  Die  Ophthalmo-Reaktion  zur  Erkennung  der  Tuberkulose  bei 

Rindern.    Inau.  Diss.,  Bern.,  1910. 
si  Melvin,  A.  D. :  The  Sixth  International  Congress  on  Tuberculosis,  vol.  4,  no. 

2,  section  7,  p.  505. 
32 Twenty-eighth  Ann.  Rept.  U.  S.  Bureau  of  Animal  Industry,  1911, 

p.  59. 

33  Meyer,  K.  F.:    The  Conjunctival  Reaction  for  Glanders.     Journ.  Infectious 

Diseases,  vol.  12,  no.  2  (March,  1913),  p.  172. 

34  Moore,  V.  A.:   Bovine  Tuberculosis  and  Its  Control.      (1913)    Published  by 

Carpenter  &  Co.,  Ithaca,  N.  Y. 

35  Moussu,   G.,  and  Mantoux,   C. :    On   the   Intradermal   Reaction   in   Animals. 

Comp.  Rend.  Acad.  Sci.  Paris,  vol.  17  (1908),  no.  11,  pp.  502-504.  Abs. 
in  Expt.  Sta.  Record,  vol.  21,  no.  2  (August,  1909). 

36  Moussu,  G. :  Sur  l'Intra-Dermo-Reaction  a  la  tuberculin.     Bull.  Soc.  Cent,  de 

Med.  Veterinaire,  vol.  63,  pp.  115-121. 

37  Moussu,  G.,  and  Mantoux,  C. :   Sur  l'Intra-Dermo-Reaction  a  la  Tuberculine 

chez.  les  Animaux.  Proceedings  6th  International  Congress  on  Tuber- 
culosis, vol.  4,  part  2,  pp.  821-835. 

ss  Moussu,  G.:  The  Intradermal  Reaction  to  Tuberculin.  Bull.  Soc.  Cent,  de  Med. 
Vet.,  vol.  85  (1908),  no.  20,  pp.  500-518;  no.  24,  pp.  649-655;  Abs.  in  Bull. 
Inst.  Pasteur,  vol.  7  (1909),  no.  4,  pp.  154-155.  Abs.  in  Expt.  Sta.  Record, 
vol.  21,  no.  6  (November,  1909). 

39  Norgaard,  V.  A.,  and  Case,  L.  N.:  The  Intradermal  Tuberculin  Test.    Biennial 

Report  of  Bd.  of  Agr.  and  Forestry  of  Hawaii  (December,  1912),  pp.  179- 

182. 
•to  Ostertag,   R. :    Die   Bekampfung   der   Tuberkulose    des    Rindes    (R.    Schoetz, 

Berlin,  1913),  pp.  216-223. 
4i  Porter,  F.  W.:  The  Intradermal  Test  for  Tuberculosis.     Amer.  Vet.  Rev.,  vol. 

41,  no.  4,  (July,  1912),  pp.  463. 

42  Raebiger  and  Rautman  :  Citation  by  Ostertag,  see  ref.  40. 

43  Romer  and  Joseph  :  Tuberculosis  and  the  Tuberculin  Reaction.     Beitr.  Klinik 

Tuberkulose,  vol.  17  (1910),  no.  2,  pp.  427-460,  pis.  8;  Abs.  in  Zeitschr. 
Immunitatsf.  u.  Expt.  Ther.,  II  Ref.,  vol.  3  (1910),  no.  8,  p.  858.  Abs.  in 
Expt.  Sta.  Record,  vol.  26,  no.  2  (February,  1912),  p.  180. 

43  Romer  and  Joseph  :   Tuberculosis  and  the  Tuberculin  Reaction.    Beitr.  Klinik. 

Tuberkulose,  vol.  17   (1910),  no.  3,  pp.  427-460,  pis.  8;  Abs.  in  Zeitschr. 

44  Romer:   Zur  Verwertung  der  intrakutanen  Reaktion  aus  Tuberkulin.     Beitr. 

Klin.  Tuberk.,  1909,  vol.  14,  no.  1. 

45  Schnurer,  J.:    Die  Diagnose  der  ansteckenden   Tierkrankheiten  mittels   der 

neueren  Immunitatsreaktionen.  Proceedings  9th  Inter.  Veterinary  Con- 
gress at  Hague,  1909. 

46  Sheldon,  S.:  Editorial,  Amer.  Journ.  Vet.  Med.,  vol.  7,  no.  7  (July,  1912). 
Bovine  Tuberculosis.     Bulletin  Missouri  State  Board  of  Agriculture, 

Columbia,  Missouri,  vol.  10,  no.  11. 

47  Titze,  C:  Arbeiten  aus  dem  Kaiserlichen  Gesundheitsamte,  1913,  vol.  43,  pp. 

505-519. 


BULLETIN  243]  INTRADERMAL  TEST  FOR  TUBERCULOSIS  145 

48  Vallee,   Declaire  and   Herbert:    The   Intradermal   Eeaction   to    Tuberculin. 

Bull.  Soc.  Cent.  Med.  Vet.,  vol.  63    (1909),  no.  6,  pp.   107-115.     Abs.   in 
Expt.  Sta.  Eecord,  vol.  22,  no.  1   (January,  1901). 

49  Vallee,  H.:  Kutane  und  Konjunctival  Tuberkulinreaktion  beim  Einde.    Hand- 

buch  der  Technik  u.  Methodik  der  Immunitatsforchung.  (1911),  pp.  382. 
so  Ward,  A.  E.,  and  Baker,  G.  S.:  The  Intradermal  Test.    Proceedings  American 

Veterinary  Medical  Association,  1910. 
si Experiments    with    Intradermal    Tests    for    Tuberculosis    in    Cattle. 

Amer.  Vet.  Eev.,  vol.  38   (1910),  no.  2,  pp.  184-193.     Abs.  in  Expt.  Sta. 

Eecord,  vol.  25,  no.  6  (October,  1911). 

52  Whitehouse,  A.  W.:  The  Intradermal  Tuberculin  Test.    Pacific  Dairy  Eeview, 

vol.  17,  no.  20,  p.  20. 

53  Wilkinson,  W.  D.:  The  Etiological  Diagnosis  of  Tuberculosis.     Brit.  Journ. 

Tuberculosis,  vol.  4  (1910),  no.  1,  pp.  40-41.     Abs.  in  Expt.  Sta.  Eecord, 
vol.  26,  no.  1  (January,  1912),  p.  85. 

54  Wills,  J.  G.:  Delayed  Eeactions  to  Tuberculin.     Eeport  of  U.  S.  Live  Stock 

Sanitary  Association,  December,  1913. 

55  Zschocke,  A.:  Die  Intrakutan-Eeaktion  bei  Tuberkulose  von  Eind  und  Schwein. 

Inau.  Diss.,  Leipzig,  1909. 
56 Die   Intrakutan-Eeaktion   bei    Tuberkulose   von    Eind    und    Schwein. 

Deutsch  Tierarztlich.  Wochensch.  (1906),  p.  28. 
57  Zwick    and    Titze,    C. :    Handbuch    der   pathogenen    Mikroorganismen    (Jena, 

1912),  2nd  ed.,  vol.  5,  pp.  711. 


1-1:6  UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


ACKNOWLEDGMENT 

I  desire  to  acknowledge  our  indebtedness  to  the  other  members  of 
the  Veterinary  Division  of  this  Experiment  Station,  all  of  whom  have 
given  assistance  in  the  work  herein  described,  and  to  Dr.  Roadhouse 
in  particular,  who  made  230  of  the  simultaneous  tests  described  in 
Table  6.  Mr.  C.  M.  Twining,  of  the  Cutter  Laboratory,  Berkeley. 
California,  has  also  rendered  considerable  assistance. 

C.  M.  II. 


Bulletin  243] 


INTRADERMAL  TEST  FOR  TUBERCULOSIS 


147 


PLATE   1 


Figures  1  and  2 

Either  of  the  two  syringes  shown  above  is  suitable  for 
intradermal  injecting.  The  shorter  one  is  a  dental  syringe 
of  30  minims  capacity,  with  a  burr  on  the  piston  which  can  be 
used  to  regulate  the  dose.  For  intradermal  work  the  needle 
point  should  be  about  one-quarter  of  an  inch  long. 


H 

i  &\#\  ,        1  ,2i  ,  !     •   ;             1 

Figure  3 


Calipers  similar  to  those  shown  above  are  useful  in  determining 
the  exact  size  reaction  of  swelling 


148 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


PLATE   2 


In  making  an  intradermal 
injection,  one  of  the  folds  of 
skin  beneath  the  base  of  the 
tail  is  grasped  between  the 
thumb  and  fingers  and  the 
needle  inserted,  slightly  hor- 
izontally, into  the  thickness  of 
the  skin  between  the  thumb 
and  fingers.  If  the  needle  is 
properly  placed  the  tuberculin 
can  be  felt  in  the  deeper  layers 
of  the  skin  as  it  is  injected, 
and  after  the  removal  of  the 


Figure  1 


needle  the  tuberculin  can  be 
felt  as  a  lump  about  the  size 
of  a  small  pea.  The  best  re- 
actions are  obtained  when  the 
tuberculin  is  injected  into  the 
deeper  layers  of  the  skin.  In- 
ject deep  enough  so  that  the 
fluid  does  not  swell  out  the 
skin  like  a  blister.  We  con- 
sider Figure  1  a  more  con- 
venient position  for  operating 
than  Figure  2. 


Figure  2 


Bulletin  243] 


INTRADERMAL  TEST  FOR  TUBERCULOSIS 


149 


PLATE  3 


The  figures  in  this  plate 
show  typical  intradermal  re- 
actions. A  reaction  to  the 
intradermal  tests  is  indicated 
by  a  swelling  or  a  thickening 
of  the  skin  at  the  point  of  in- 
jection. The  swelling  may  be 
either  soft  or  pendulous,  as 
in  Figure  1,  or  hard  and  in- 
flamed as  in  Figure  2. 


Figure  2 

In  some  instances  we  have 
observed  that  the  early  local 
reactions  disappear  by  the 
forty-eighth  hour,  while  in 
other  cows  delayed  reactions 
have  been  noted  that  did  not 
appear  until  the  ninetieth 
hour.  In  order  to  be  certain 
of  every  case,  observations 
should  be  taken  about  the 
thirtieth  and  seventieth  hours 
after  injection. 


Figure  1 

A  recognizable  reaction 
may  be  present  at  the  sixth 
hour,  but  generally  the  re- 
action swelling  is  first  clear 
about  the  twelfth  hour  and 
continues  to  increase  in  size 
until    the    forty-eighth    hour. 


Figure  3 


150 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT.  STATION 


PLATE   4 


Figure  1 

should  depend  on  the  shape, 
appearance,  tenseness,  sensi- 
tiveness and  location  with  re- 
spect to  the  exact  point  of  in- 


The  use  of  calipers  to  de- 
termine the  exact  size  of  the 
swellings  is  shown  in  the  fig- 
ures on  this  plate.  However, 
in  judging  a  local  swelling  to 
determine  if  it  is  a  positive 
reaction    to    tuberculin,    one 


Figure  3 


Figure  2 

jection  more  than  upon  the 
actual  measurements.  Only 
experience  can  teach  one  how 
to  be  certain  of  a  positive  re- 
action when  the  local  swelling 
is  small.  This  is  especially 
true  when  tuberculin  contain- 
ing glycerin  is  used. 


Bulletin  243] 


INTRADERMAL  TEST  FOR  TUBERCULOSIS 


151 


PLATE  5 


As  shown  in  Figure  1  the  intra- 
dermal reaction  is  sometimes  so 
large  that  it  can  be  seen  without 
raising  the  tail. 

Figure  2  shows  an  intradermal 
reaction  that  obliterates  both  sub- 
caudal  folds. 


Figure  1 

Figure  3  shows  the  swellings 
due  to  two  injections  with  a  six- 
day  interval.  The  left  subcaudal 
fold  was  injected  July  30th  and  a 
local  reaction  the  size  of  an  orange 


Figure 


occurred,  which  persisted  sev- 
eral weeks.  The  right  fold 
was  injected  August  5th  and 
resulted  in  a  local  reaction  the 
size  of  a  walnut.  (See  Animal 
1,  Table  6.)  Thermal  reac- 
tions usually  accompany  in- 
tradermal reactions. 

One  advantage  of  the  in- 
tradermal reaction  is  that  it 
often  leaves  a  more  or  less 
permanent  mark  which  can  be 
used  to  identify  the  reacting 
animals  for  several  weeks. 


Figure  3 


152 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT  STATION 


PLATE   6 


Figure  1 


Figure  1  is  not  a  cut  of  a  re- 
action, but  shows  a  swelling  caused 
by  the  application  of  a  strong 
solution  of  lysol.  Irritating  disin- 
fectants should  not  be  used  before 
applying  the  intradermal  test. 


Figure  2  shows  an  intradermal 
reaction  in  a  calf  six  weeks  of  age. 
The  test  is  especially  adapted  to  the 
testing  of  young  calves  and  wild 
range  cattle. 


Figure 


Bulletin  243] 


INTRADERMAL  TEST  FOR  TUBERCULOSIS 


153 


PLATE  7 


The  ophthalmic  or 
eye  test  with  tuberculin 
may  equal  the  other 
forms  of  tuberculin  test- 
ing in  accuracy,  but 
under  the  average  out- 
door conditions  of  Cali- 
fornia is  not  as  suitable 
as  the  intradermal  test. 

Figure  1  shows  a 
mild  ophthalmic  reaction 
twelve  hours  after  injec- 
tion. 


Figure  1 


Figure  2  shows  a 
severe  ophthmalic  reac- 
tion twelve  hours  after 
injection. 


Figure  2 


154 


UNIVERSITY  OF  CALIFORNIA EXPERIMENT   STATION 


PLATE  8 


Figure  1 


Figure  2 


Figure  3 


The  intradermal  test  is  especially  adapted  to  the  testing  of  swine. 
Injections  may  be  made  into  the  back  of  the  ear  near  its  base  as  in 
Figure  1,  or  into  the  edge  of  the  ear  as  in  Figure  2.  The  reaction 
swellings  are  similar  to  those  in  cattle  (see  Figure  3). 


STATION  PUBLICATIONS  AVAILABLE  FOR  DISTRIBUTION 


REPORTS 

1897.      Resistant  Vines,  their  Selection,  Adaptation,  and  Grafting.     Appendix  to  Viticultural 
Report  for  1896. 

1902.  Report  of  the  Agricultural  Experiment   Station  for   1898-1901. 

1903.  Report  of  the  Agricultural   Experiment   Station  for   1901-03. 

1904.  Twenty-second  Report  of  the  Agricultural  Experiment  Station  for  1903-04. 


BULLETINS 


No. 

128.  Nature,  Value,  and  Utilization  of 
Alkali  Lands,  and  Tolerance  of 
Alkali.  (Revised  and  Reprint, 
1905.) 

133.  Tolerance  of  Alkali  by  Various  Cul- 
tures. 

168.  Observations  on   Some  Vine  Diseases 

in  Sonoma  County. 

169.  Tolerance    of    the     Sugar     Beet     for 

Alkali. 

170.  Studies  in  Grasshopper  Control. 

171.  Commercial    Fertilizers.       (June    30, 

1905.) 
174.   A  New  Wine-Cooling  Machine. 

177.  A   New   Method  of  Making   Dry   Red 

Wine. 

178.  Mosquito   Control. 

179.  Commercial  Fertilizers.    (June,  1906.) 

182.  Analysis    of    Paris    Green    and    Lead 

Arsenic.    Proposed  Insecticide  Law. 

183.  The  California  Tussock-moth. 

184.  Report    of    the    Plant    Pathologist    to 

July  1,  1906. 

185.  Report  of  Progress  in  Cereal  Investi- 

gations. 

186.  The  Oidium  of  the  Vine. 

187.  Commercial  Fertilizers.     (Jan.,  1907.) 
189.   Commercial  Fertilizers.    (June,  1907.) 

194.  Commercial  Fertilizers.     (Dec,  1907.) 

195.  The  California   Grape  Root-worm. 

197.  Grape     Culture     in     California;     Im- 

proved   Methods    of    Wine-making; 
Yeast  from  California  Grapes. 

198.  The  Grape  Leaf-Hopper. 

199.  Bovine  Tuberculosis. 

201.  Commercial  Fertilizers.    (June,  1908.) 

202.  Commercial  Fertilizers.     (Dec,  1908.) 


No. 
203. 

204. 

205. 
206. 
207. 
208. 
209. 
211. 

212. 
213. 
215. 

216. 


217. 
220. 

222. 
223. 
224. 
225. 
226. 
227. 
230. 
234. 

237. 

240. 
241. 
242. 
243. 

244. 


Report    of    the    Plant    Pathologist    to 

July  1,   1909. 
The    Dairy    Cow's    Record    and    the 

Stable. 
Commercial  Fertilizers.     (Dec,  1909.) 
Commercial  Fertilizers.    (June,  1910.) 
The  Control  of  the  Argentine  Ant. 
The  Late  Blight  of  Celery. 
The  Cream  Supply. 
How  to  Increase  the  Yield  of  Wheat 
in    California. 
California  White  Wheats. 
The  Principles  of  Wine-making. 
The    House    Fly    in    its    Relation    to 

Public  Health. 
A    Progress    Report    upon    Soil    and 

Climatic     Factors     Influencing     the 

Composition  of  Wheat. 
Honey   Plants   of   California. 
Fumigation    Studies    No.    5 ;    Dosage 

Tables. 
The  Red  or  Orange  Scale. 
The  Black  Scale. 

The  Production  of  the  Lima  Bean. 
Tolerance  of  Eucalyptus  for  Alkali. 
The  Purple  Scale. 
Grape  Vinegar. 
Enological  Investigations. 
Red     Spiders     and    Mites    of     Citrus 

Trees. 
Pork     Production     under     California 

Conditions. 
Commercial   Fertilizers. 
Vine   Pruning  in   California. 
Humus  in  California  Soils. 
The     Intradermal     Test     for     Tuber- 
culosis in  Cattle  and  Hogs. 
Utilization   of  Waste   Oranges. 


CIRCULARS 


No. 
29. 


4  6. 
52. 

61. 

62. 

65. 

66. 
68. 
69. 
70. 


Preliminary  Announcement  Concern- 
ing Instruction  in  Practical  Agri- 
culture upon  the  University  Farm, 
Davis,  Cal. 

Suggestions  for  Garden  Work  in  Cali- 
fornia Schools. 

Information  for  Students  Concerning 
the  College  of  Agriculture. 

University  Farm  School. 

The  School  Garden  in  the  Course  of 
Study. 

The  California   Insecticide  Law. 

Insecticides  and   Insect   Control. 

The  Prevention  of  Hog  Cholera. 

The  Extermination  of  Morning-Glory. 

Observation  of  the  Status  of  Corn 
Growing  in  California. 


No. 

74. 
75. 
76. 

77. 
78. 

79. 
80. 
82. 

83. 
84. 
85. 


87. 


Rice. 

A  New  Leakage  Gauge. 

Hot  Room  Callusing. 

University  Farm  School. 

Announcement  of  Farmers'  Short 
Courses  for   1912. 

List  of  Insecticide  Dealers. 

Boys'  and  Girls'  Clubs. 

The  Common  Ground  Squirrels  of 
California. 

Potato   Growing  Clubs. 

Mushrooms  and  Toadstools. 

A  Preliminary  Report  of  a  Nematode 
Observed  on  Citrus  Roots  and  its 
Possible  Relation  with  the  Mottled 
Appearance  of  Citrus   Trees. 

Alfalfa. 


CIRCULARS—  (Continued) 


No. 

88.  Advantages   to   the   Breeder   in   Test- 

ing   his    Pure-bred    Cows    for    the 
Register  of  Merit. 

89.  Hog  Cholera  and  its  Prevention. 

90.  Tuberculosis  in  Cattle  and  Hogs. 

91.  Disinfection  on  the  Farm. 

92.  Infectious   Abortion    and    Sterility    in 

Cows. 

98.  Plowing     and     Cultivating     Soils     in 

California. 

99.  Hatching  and  Rearing  of  Chicks. 

100.  Pruning  Frosted  Citrus  Trees. 

101.  Codling   Moth   Control   in   the    Sacra- 

mento Valley. 


No. 
102.  The  Woolly  Aphis. 

106.  Directions  for  Using  Anti-Hog-Cholera 

Serum. 

107.  Spraying    Walnut    Trees    for    Blight 

and  Aphis  Control. 

108.  Grape  Juice. 

109.  Community  or  Local  Extension  Work 

by    the    High    School    Agricultural 
Department. 

110.  Green  Manuring  in  California. 

111.  The    Use    of    Lime    and    Gypsum    on 

California   Soils. 

112.  The  County  Farm  Adviser. 

113.  Announcement    of    Correspondence 

Courses  in  Agriculture. 

114.  Increasing  the  Duty  of  Water. 

115.  Grafting  Vinifera  Vineyards. 

116.  Silk  Worm  Experiments. 


